Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof

ABSTRACT

A highly efficient method for generating human antibodies in particular which are specific to be RSV fusion protein which combines in vitro primary of human spleen cells and antigen boosting in SCID mice is taught. This method provides for very high human antibody titers which are predominantly of the IgG isotype which contain antibodies of high specificity and affinity to desired antigens. This method is well suited for generating human monoclonal antibodies for therapeutic and diagnostic applications as well as for rescue of human cells for generation of combinational human antibody gene libraries. Two human monoclonal antibodies, RF-1 and RF-2 which each possess an affinity for RSV F-protein ≦2×10 −9  Molar are taught as well as their corresponding amino acid and DNA sequences. These antibodies are to be used therapeutically and prophylactically for treating or preventing RSV infection, as well as for diagnosis of RSV in analytes.

BACKGROUND OF THE INVENTION

[0001] Respiratory syncytial virus (RSV) is a Parmixovirus of thePneumovirus genus which commonly infects the upper and lower respiratorytract. It is so contagious that by age two, a large percentage ofchildren have been infected by it. Moreover, by age four, virtually allhumans have an immunity to RSV.

[0002] Typically, RSV infections are mild, remaining localized in theupper respiratory tract and causing symptoms similar to a common coldwhich require no extensive treatment. However, in some subjects, e.g.,immunosuppressed individuals such as infants, elderly persons orpatients with underlying cardiopulmonary diseases, the virus maypenetrate to the lower respiratory tract requiring hospitalization andbreathing support. In some of these cases, RSV infection may causepermanent lung damage or even be life threatening. In the United Statesalone, RSV results in about 90,000 hospitalizations each year, andresults in about 4500 deaths.

[0003] RSV appears in two major strain subgroups, A and B, primarilybased on serological differences associated with the attachmentglycoprotein, G. The major surface glycoprotein, i.e., the 90 kD Gprotein, can differ up to 50% at the amino acid level between isolatesJohnson et al, Proc. Natl. Acad. Sci. (1987), 84, 5625-5629. Bycontrast, a potential therapeutic target, the 70 kD fusion (F) protein,is highly conserved across different RSV strains, about i.e., 89% on theamino acid level Johnson et al, J. Gen. Virol. (1988), 69, 2623-2628,Johnson et al, J. Virol. (1987), 10, 3163-3166, P. L. Collins. PlenumPress, NY (1991), 103-162. Moreover, it is known that antibodieselicited against F-protein of a given type are cross-reactive with theother type.

[0004] The F-protein is a heterodimer, generated from a linearprecursor, consisting of disulfide-linked fragments of 48 and 23 kDrespectively Walsh et al, J. Gen. Virol, (1985), 66, 401-415. Inhibitionof syncytia formation by polyclonal antibodies is associated withsignificant reaction to the 23 kD fragment.

[0005] As noted, while RSV infections are usually mild, in someindividuals RSV infections may be life threatening. Currently, severeRSV infection is treated by administration of the antiviral agentRibavarin. However, while Ribavarin exhibits some efficacy incontrolling RSV infection, its use is disfavored for several reasons.For example, it is highly expensive and may be administered only inhospitals. Other known RSV treatments only treat the symptoms of RSVinfection and include the use of aerosolized bronchodilators in patientswith bronchiolitis and corticosteroid therapy in patients withbronchiolitis and RSV pneumoma.

[0006] To date, RSV vaccines intended to boost antiviral protectiveantibodies have been largely unsuccessful. For example, a vaccine basedon formalin-inactivated RSV that was tested approximately 25 years ago,induced antibodies that were deficient in fusion inhibiting activityMurphy et al, Clinical Microbiology (1988), 26, 1595-1597, and sometimeseven exacerbated the disease. This may potentially be explained to theinability of the formalin inactivated virus to induce protectiveantibodies. While high antibody titers were measured in vaccinerecipients, specific protective titers were lower than in the controlpopulation. This may be because formalin inactivated RSV does notdisplay the necessary conformational epitopes required to elicitprotective antibodies.

[0007] While there is no known effective RSV vaccine to date, thereexists some clinical evidence that antibody therapy may conferprotection against RSV infection in susceptible individuals, and mayeven clear an existing RSV infection. For example, it has been reportedthat newborn infants show a low incidence of severe bronchiolitis, whichis hypothesized to be attributable to the presence of protectivematernal antibodies Ogilvie et al J. Med Virol (1981), 7, 263-271. Also,children who are immune to reinfection exhibit statistically higheranti-F-protein titers than those who are reinfected. Moreover,intravenous immune globulin (IVIG) prepared from high titer RSV-immunedonors reduces nasal RSV shedding and improves oxygenation Hemming etal, Anti. Viral Agents and Chemotherapy (1987), 31, 1882-1886. Also,recent studies have suggested that the virus can be fought and lungdamage prevented by administering RSV-enriched immune globulin (RSVIG)Groothuis et al, The New England J. Med. (1993), 329, 1524-1530, K.McIntosh. The New England J. Med. (1993), 329, 1572-1573, J. R.Groothuis. Antiviral Research, (1994), 23, 1-10, Siber et al, J.Infectious Diseases (1994), 169, 1368-1373, Siber et al, J. InfectiousDiseases (1992), 165:456-463.

[0008] Similarly, some animal studies suggest that antibody therapy withvirus neutralizing antibodies may confer protection against RSV or evenclear an existing RSV infection. For example, in vitro neutralizingmouse monoclonal antibodies have been reported to protect mice againstinfection and also to clear established RSV infections Taylor et al, J.Immunology, (1984), 52, 137-142, Stott et al., “Immune Responses. VirusInfections and Disease, I.R.L. Press, London (1989), 85-104. Also,monoclonal antibodies to the F-protein of RSV have shown high efficacyin both in vitro and in vivo RSV models Tempest et al, Bio/Technology,(1991), 9, 266-271, Crowe et al, Proc. Natl. Acad. Sci. (1994), 91,1386-1390, Walsh et al, Infection and Immunity, (1984), 43, 756-758,Barbas III. et al. Proc. Natl. Acad. Sci. (1992), 89, 10164-10168,Walsh. et al, J. Gen. Virol. (1986), 67, 505-513. Antibodyconcentrations as low as 520-2000 μg/kg body weight have been reportedto result in almost instant recovery in animal studies Crowe et al,Proc. Natl. Acad. Sci. (1994), 91, 1386-1390. Moreover, these monoclonalantibodies have been disclosed to neutralize both A and B strains,including laboratory strains and wildtype strains. These antibodies wereadministered either by injection Groothuis et al, The New England J.Med. (1993), 329, 1524-1530, Siber et al, J. Infectious Diseases (1994),169, 1368-1373 or by aerosol Crowe et al, Proc. Natl. Acad. Sci. (1994),91, 1386-1390.

[0009] Two different types of potentially therapeutic monoclonalantibodies to the RSV F-protein have been previously described in theliterature, humanized murine antibodies Tempest et al, Biol. Technology,(1991) 9, 266-271, or true human antibodies (Fab fragments) Barbas III,et al, Proc. Natl. Acad. Sci. (1992), 89, 10164-10168. Humanized murineantibodies were generated by CDR grafting a cross-strain neutralizingmurine anti-F-protein antibody onto a generic human Fc, as well asstructural areas of the variable part. The human Fab fragments wereproduced by combinatorial library technology using human bone marrowcells obtained from an HIV positive donor (immunocompromised). Thetherapeutic in vivo titers of the humanized and human RSV antibodieswere 5 and 2 mg/kg body weight, respectively. It is noted, however, thatthe humanized antibodies were tested in a syncytia inhibition assay,whereas the human anti-RSV Fab fragments were assayed to determine theirvirus neutralization activity. Therefore, the results reported with thehumanized and human anti-RSV antibodies are not directly comparable.

[0010] The Fab fragment generated by the combinatorial librarytechnology were disclosed to be efficient in aerosol. This is probablybecause of the relatively small size of the molecule. These results arehighly encouraging because a major target population for an RSV vaccineis infants. Therefore, aerosol is a particularly desirable mode ofadministration.

[0011] However, notwithstanding the previous published reports ofhumanized and Fab fragments specific to RSV, there still exists asignificant need for improved anti-RSV antibodies having improvedtherapeutic potential, in particular anti-RSV antibodies which possesshigh affinity and specificity for the RSV F-protein which effectivelyneutralize and prevent RSV infection.

[0012] Antibody therapy can be subdivided into two principally differentactivities: (i) passive immunotherapy using intact non-labeledantibodies or labeled antibodies and (ii) active immunotherapy usinganti-idiotypes for re-establishment of network balance in autoimmunity.

[0013] In passive immunotherapy, naked antibodies are administered toneutralize an antigen or to direct effector functions to targetedmembrane associated antigens. Neutralization would be of a lymphokine, ahormone, or an anaphylatoxin, i.e., C5a. Effector functions includecomplement fixation, macrophage activation and recruitment, and antibodydependent cell mediated cytotoxicity (ADCC). Naked antibodies have beenused to treat leukemia Ritz et al. S.F. Blood, (1981), 58, 141-152 andantibodies to GD2 have been used in treatments of neuroblastomas Schulzet al. Cancer Res. (1984), 44:5914 and melanomas Irie et al., Proc.Natl. Acad. Sci., (1986, 83:8694. Also, intravenous immune gammaglobulin (IVIG) antibodies with high anti-RSV titers recently were usedin experimental trials to treat respiratory distress caused by RSVinfection Hemming et al, Anti. Viral Agents and Chemotherapy, (1987),31, 1882-1886, Groothuis et al., The New England J. Med. (1993), 329,1524-1530, K. McIntosh. The New England J. Med. (1993), 329, 1572-1573,J. R. Groothuis, Antiviral Research, (1994), 23, 1-10, Siber et al, J.Infectious Diseases (1994), 169, 1368-1373.

[0014] The therapeutic efficacy of a monoclonal antibody depends onfactors including, e.g., the amount, reactivity, specificity and classof the antibody bound to the antigen. Also, the in vivo half-life of theantibody is a significant therapeutic factor.

[0015] Still another factor which may significantly affect thetherapeutic potential of antibodies is their species of origin.Currently, monoclonal antibodies used for immunotherapy are almostexclusively of rodent origin Schulz et al. Cancer Res. (1984), 44:5914,Miller et al, Blood (1981), 58, 78-86, Lanzavecchia et al, J. Edp. Med.(1988), 167, 345-352, Sikora et al, Br. Med. Bull. (1984), 40:240,Tsujisaki et al, Cancer Research (1991), 51:2599, largely because thegeneration of rodent monoclonal antibodies uses well characterized andhighly efficient techniques Köhler et al, Nature, (1975), 256:495,Galfre et al. Nature, (1977), 266:550. However, while rodent monoclonalantibodies possess therapeutic efficacy, they can present restrictionsand disadvantages relative to human antibodies. For example, they ofteninduce sub-optimal stimulation of host effector functions (CDCC, ADCC,etc.). Also, murine antibodies may induce human anti-murine antibody(HAMA) responses Schroff et al, Can. Res. (1985, 45:879-885, Shawler etal, J. Immunol. (1985), 135:1530-1535. This may result in shortenedantibody half-life Dillman et al, Mod. (1986), 5, 73-84, Miller et al.Blood, (1983), 62:988-995 and in some instances may cause toxic sideeffects such as serum sickness and anaphylaxis.

[0016] In some subjects, e.g., heavily immunosuppressed subjects (e.g.,patients subjected to heavy chemical or radiation mediated cancertherapy Irie et al, Proc. Natl. Acad. Sci. (1986), 83:8694, Dillman etal, Mod. (1986), 5, 73-84, Koprowski et al, Proc. Natl. Acad. Sci.(1984), 81:216-219), use of murine monoclonal antibodies causes limitednegative side effects. By contrast, in patients with normal orhyperactive immune systems, murine antibodies, at least for some diseaseconditions may exhibit limited efficacy.

[0017] In an effort to obviate limitations of murine monoclonalantibodies, recombinant DNA techniques have been applied to producechimeric antibodies Morrison et al, Proc. Natl. Acad. Sci. (1984),81:216-219, Boulianne et al, Nature, (1984), 312, 644-646, humanizedantibodies by “CDR grafting” Riechmann et al. Nature (1984), 332,323-327 and “veneered” antibodies by substitution of specific surfaceresidues with other amino acids to alleviate or eliminate antigenicity.

[0018] However, although such antibodies have been used successfullyclinically Gillis et al, J. Immunol. Meth (1989), 25:191, they haveproven cumbersome to produce. This is because the understanding of therequirements for optimal antigen recognition and affinity is not yetfully understood. Also, the human framework and the mouse CDR regionsoften interact sterically with a negative effect on antibody activity.Moreover, such antibodies sometimes still induce strong HAMA responsesin patients.

[0019] Human antibodies present major advantages over their murinecounterparts; they induce optional effector functions, they do notinduce HAMA responses and host antigen-specific antibodies may lead toidentification of epitopes of therapeutic value that may be too subtleto be recognized by a xenogeneic immune system Lennox et al. “MonoclonalAntibodies in Clinical Medicine.” London: Academic Press (1982).

[0020] While human antibodies are highly desirable, their production iscomplicated by various factors including ethical considerations, and thefact that conventional methods for producing human antibodies are ofteninefficient. For example, human subjects cannot generally be adequatelyimmunized with most antigens because of ethical and safetyconsiderations. Consequently, reports of isolation of human monoclonalantibodies with useful affinities, ≧10⁸ molar to specific antigens arefew McCabe et al, Cancer Research, (1988), 48, 4348-4353. Also,isolation of anti-viral human monoclonal antibodies from donor primedcells has proved to be unwieldy. For example, Gorny reported that only 7of 14,329 EBV transformed cultures of peripheral blood mononuclear cells(PMBC's) from HIV positive donors resulted in stable, specific anti-HIVantibody producing cell lines Gorny et al. Proc. Natl. Acad. Sci.(1989), 86:1624-1628.

[0021] To date, most human anti-tumor antibodies have been generatedfrom peripheral blood lymphocytes (PBLs) Irie et al, Br. J. Cancer,(1981), 44:262 or tumor draining lymph node lymphocytes Schlom et al,Proc. Natl. Acad. Sci. (1980), 77:6841-6845, Cote et al, Proc. Natl.Acad. Sci. (1983), 80:2026-2030 from cancer patients. However, suchantibodies often react with intracellular, and thus therapeuticallyuseless antigens Ho et al, In Hybridoma Technology, Amsterdam (1988),37-57 or are of the IgM class McCabe et al, Cancer Research (1988), 48,4348-4353, a class of antibodies with lesser ability to penetrate solidtumors than IgGs. Few of these human antibodies have moved to clinicaltrials Drobyski et al, R.C. Transplantation (1991), 51, 1190-1196,suggesting that the rescued antibodies may possess sub-optimalqualities. Moreover, since these approaches exploit the testing donorprimed B cells, it is clear that these cells are not an optimal sourcefor rescue of useful monoclonal antibodies.

[0022] Recently, generation of human antibodies from primed donors hasbeen improved by stimulation with CD40 resulting in expansion of human Bcells Banchereau et al, F. Science (1991), 251:70, Zhang et al, J.Immunol. (1990), 144, 2955-2960, Tohma et al, J. Immunol. (1991),146:2544-2552 or by an extra in vitro booster step primer toimmortalization Chaudhuri et al, Cancer Supplement (1994), 73,1098-1104. This principle has been exploited to generate humanmonoclonal antibodies to Cytomegalovirus, Epstein-Barr Virus (EBV) andHemophilus influenza with cells from primed donors (42-44), with asignificantly higher yield than obtained with other methods (32).

[0023] Moreover, to address the limitations of donor priming,immunization and cultivation ex vivo of lymphocytes from healthy donorshas been reported. Some success in generating human monoclonalantibodies using ex homine boosting of PBL cells from primed donors hasbeen reported Maeda et al, Hybridoma (1986), 5:33-41, Kozbor et al, J.Immunol. (1984), 14:23, Duchosal et al, Nature (1992, 355:258-262. Thefeasibility of immunizing in vitro was first demonstrated in 1967 byMishell and Dutton Mishell et al, J. Exp. Med (1967), 126:423-442 usingmurine lymphocytes. In 1973, Hoffman successfully immunized humanlymphocytes Hoffman et al, Nature (1973), 243:408-410. Also, successfulprimary immunizations have been reported with lymphocytes fromperipheral blood Luzzati et al. J. Exp. Med. (1975), 144:573:585, Misitiet al, J. Exp. Med. (1981), 154:1069-1084, Komatsu et al. Int. Archs.Allergy Appl. Immunol. (1986), 80:431-434, Ohlin et al, C.A.K.Immunology (1989), 68:325 (1989) tonsils Strike et al, J. Immunol.(1978), 132:1789-1803 and spleens, the latter obtained from trauma Ho etal, In Hybridoma Technology, Amsterdam (1988), 37-57, Boerner et al, J.Immunol. (1991), 147:86-95, Ho et al, J. Immunol. (1985), 135:3831-3838,Wasserman et al, J. Immunol. Meth. (1986), 93:275-283, Wasserman et al,J. Immunol. Meth. (1986), 93:275-283, Brams et al, Hum. Antibod.Hybridomas (1993), 4, 47-56, Brams et al. Hum. Antibod. Hybridomas(1993), 4, 57-65 and idiopathic thrombocytopenia purpura (ITP) patientsBoerner et al, J. Immunol. (1991), 147:86-95, Brams et al, Hum. Antibod.Hybridomas (1993) 4, 47-56, Brams et al, Hum. Antibod. Hybridomas(1993), 4, 5765, McRoberts et al, “In Vitro Immunization in HybridomaTechnology”, Elsevier, Amsterdam (1988), 267-275, Lu et al, P. Hybridoma(1993), 12, 381-389.

[0024] In vitro immunization offers considerable advantages, e.g.,easily reproducible immunizations, lends itself easily to manipulationof antibody class by means of appropriate cultivation and manipulationtechniques Chaudhuri et al. Cancer Supplement (1994), 73, 1098-1104.Also, there is evidence that the in vivo tolerance to self-antigens isnot prevalent during IVI Boerner et al, J. Immunol. (1991), 147:86-95,Brams et al, J. Immunol. Methods (1987), 98:11. Therefore, thistechnique is potentially applicable for production of antibodies toself-antigens, e.g., tumor markers and receptors involved inautoimmunity.

[0025] Several groups have reported the generation of responses to avariety of antigens challenged only in vitro, e.g., tumor associatedantigens (TAAs) Boerner et al. J. Immunol. (1991), 147:86-95, Borrebaecket al, Proc. Natl. Acad. Sci. (1988), 85:3995. However, unfortunately,the resulting antibodies were typically of the IgM and not the IgGsubclass McCabe et al, Cancer Research (1988), 48, 4348-4353, Koda etal, Hum. Antibod. Hybridomas, (1990), 1:15 and secondary (IgG) responseshave only been reported with protocols using lymphocytes from immunizeddonors. Therefore, it would appear that these protocols only succeed ininducing a primary immune response but require donor immunized cells forgeneration of recall responses.

[0026] Also, research has been conducted to systematically analyzecultivation and immunization variables to develop a general protocol foreffectively inducing human monoclonal antibodies in vitro Boerner, J.Immunol. (1991) 147:86-95, Brams et al, Hum. Antibod. Hybridomas (1993),4, 47-56, Lu et al, Hybridoma (1993), 12, 381-389. This has resulted inthe isolation of human monoclonal antibodies specific for ferritinBoerner et al, J. Immunol. (1991), 147:86-95, induced by IVI of naivehuman spleen cells. Also, this research has resulted in a protocol bywhich de novo secondary (IgG) responses may be induced entirely in vitroBrains et al, Hum. Antibod. Hybridomas (1993), 4, 57-65.

[0027] However, despite the great potential advantages of IVI, theefficiency of such methods are severely restricted because of the factthat immune cells grow in monolayers in culture vessels. By contrast, invivo germinal centers possessing a three-dimensional structure are foundin the spleen during the active phases of an immune response. Thesethree-dimensional structures comprise activated T- and B-cellssurrounded by antigen-presenting cells which are believed by themajority of immunologists to compare the site of antigen-specificactivation of B-cells.

[0028] An alternative to the natural splenic environment is to“recreate” or mimic splenic conditions in an immunocompromised animalhost, such as the “Severe Combined Immune Deficient” (SCID) mouse. Humanlymphocytes are readily adopted by the SCID mouse (hu-SCID) and producehigh levels of immunoglobulins Mosier et al, Nature (1988), 335:256,McCune et al, L. Science (1988), 241, 1632-1639. Moreover, if the donorused for reconstitution has been exposed to a particular antigen, astrong secondary response to the same antigen can be elicited in suchmice. For example, Duchosal et al. Duchosal et al, Nature (1992),355:258-262 reported that human peripheral blood B-cells from a donorvaccinated with tetanus toxoid 17 years prior could be restimulated inthe SCID environment to produce high serum levels, i.e., around 10⁴.They further disclosed cloning and expression of the genes of two humananti-TT antibodies using the lambda and the M13 phage combinatoriallibrary approach Huse et al, R.A. Science (1989), 246:1275 from theextracted human cells. The reported antigen affinities of the antibodieswere in the 10⁸-10⁹/M range. However, this protocol required donorprimed cells and the yield was very low, only 2 clones were obtainedfrom a library of 370,000 clones.

[0029] Therefore, previously the hu-SPL-SCID mouse has only beenutilized for producing human monoclonal antibodies to antigens whereinthe donor has either been efficiently primed naturally or by vaccinationStähli et al, Methods in Enzymology (1983), 92, 26-36, which in mostcases involves exposure to viral or bacterial antigens. Also, thereported serum titer levels using the hu-SCID animal model aresignificantly lower than what is typically achieved by immunization ofnormal mice.

[0030] Additionally, two protocols have been described by whichinduction of primary antibody responses can be followed by induction ofsecondary antibody responses in hu-SCID mice using naive humanlymphocytes. However, use of both of these protocols are substantiallyrestricted. In the first protocol, primary responses are induced inhu-SCID mice into which human fetal liver, thymus and lymph nodes havebeen surgically implanted. However, this method is severely restrictedby the limited availability of fetal tissue, as well as the complicatedsurgical methodology of the protocol McCune et al, L. Science (1988),241, 1632-1639. In the second protocol, lethally irradiated normal micewere reconstituted with T- and B-cell depicted human bone marrow andSCID mouse bone marrow cells Lubin et al, Science, (1991), 252:427.However, this method is disadvantageous because it requires a four monthincubation period. Moreover, both protocols result in very low antibodytiters, i.e., below 10⁴.

[0031] Also, Carlson et al. Carlsson et al, J. Immunol. (1992),148:1065-1071 described in 1992 an approach using PBMCs from an antigen(tetanus toxoid) primed donor. The cells were first depleted ofmacrophages and NK cells before being subjected to a brief in vitrocultivation and priming period prior to transfer into a SCID mouse. Thehu-SPL-SCID mouse was then boosted with antigen. This method wasreported to result in average TT specific human IgG titers of

10⁴ in the hu-SPL-SCID serum, with up to 5×10⁵ reported.

[0032] Production of human monoclonal antibodies further typicallyrequires the production of immortalized B-cells, in order to obtaincells which secrete a constant, ideally permanent supply of the desiredhuman monoclonal antibodies. Immortalization of B-cells is generallyeffected by one of four approaches: (i) transformation with EBV, (ii)mouse-human heterofusion, (iii) EBV transformation followed byheterofusion, and (iv) combinatorial immunoglobulin gene librarytechniques.

[0033] EBV transformation has been used successfully in a number ofreports, mainly for the generation of anti-HIV antibodies Gorny et al,Proc. Natl. Acad. Sci. (1989), 86:1624-1628, Posner, et al, J. Immunol.(1991), 146:4325-32. The main advantage is that approximately one ofevery 200 B-cells becomes transformed. However, EBV transformed cellsare typically unstable, produce low amounts of mainly IgM antibody,clone poorly and cease making antibody after several months ofculturing. Heterofusion Carrol, et al, J. Immunol. Meth. (1986),89:61-72 is typically favored for producing hybridomas which secretehigh levels of IgG antibody. Hybridomas are also easy to clone bylimiting dilution. However, a disadvantage is the poor yield, i.e., ≦1hybridomas per 20,000 lymphocytes Boerner, et al, J. Immunol. (1991),147:86-95, Ohlin. et al, C.A.K. Immunology (1989), 68:325, Xiu-mei etal, Hum. Antibod. Hybridomas (1990), 1:42, Borrebaeck C.A.K. Abstract atthe “Second International Conference” on “Human Antibodies andHybridomas.” Apr. 26-28, 1992, Cambridge, England. Combining tBVtransformation followed by heterofusion offers two advantages: (i) humanB-cells fuse more readily to the fusion partner after EBVtransformation, and (ii) result in more stable, higher producinghybridomas Ohlin, et al, Immunology (1989), 68:325, Xiu-mei. et al, Hum.Antibod. Hybridomas

[0034] (1990), 1:42, Borrebaeck C.A.K. Absract at the “SecondInternational Conference” on “Human Antibodies and Hybridomas.” Apr.26-28, 1992, Cambridge, England. The advantage of the final technique,i.e., combinatorial immunoglobulin gene library technique is the factthat very large libraries can be screened by means of the M13 Fabexpression technology Huse, et al. Science (1989), 246:1275, WilliamHuse, Antibody Engineering: A Practical Guide, Borrebaeck C.A.K., ed.5:103-120 and that the genes can easily be transferred to a productioncell line. However, the yield is typically extremely low, on the orderof 1 per 370,000 clones Duchosal, et al. Nature (1992), 355:258-262.

[0035] Thus, based on the foregoing, it is apparent that more efficientmethods for producing human monoclonal antibodies, in particularantibodies specific to RSV, would be highly advantageous. Moreover, itis also apparent that human antibodies specific to the RSV F-proteinhaving superior binding affinity, specificity and effector functionsthan those currently available would also be highly desirable.

OBJECTS OF THE INVENTION

[0036] It is an object of the invention to provide improved methods forproducing human antibodies of high titers which are specific to desiredantigens.

[0037] It is a more specific object of the invention to provide a novelmethod for producing high titer human antibodies which comprises (i)antigen priming of naive human splenocytes in vitro, (ii) transferral ofin vitro antigen primed splenocyte cells to an immunocompromised donor,e.g., a SCID mouse, and (iii) boosting with antigen.

[0038] It is another specific object of the invention to provideimproved methods for producing human monoclonal antibodies which arespecific to respiratory syncytial virus (RSV), and in particular the RSVfusion (F) protein.

[0039] It is another object of the invention to provide an improvedmethod for producing EBV immortalized B-cells which favors the formationof EBV immortalized B-cells which predominantly secrete IgG.

[0040] It is a more specific object of the invention to provide animproved method for producing EBV immortalized human B-cells whichpredominantly secrete IgG's which comprises:

[0041] (i) antigen priming of naive human splenocytes in vitro;

[0042] (ii) transferral of such in vitro antigen primed naivesplenocytes to an immunocompromised donor, e.g., a SCID mouse;

[0043] (iii) boosting the immunocompromised donor with antigen;

[0044] (iv) isolation of human antibody producing B-cells from theantigen boosted immunocompromised donor, e.g., SCID mouse; and

[0045] (v) EBV transformation of said isolated human antibody producingB-cells.

[0046] It is another object of the invention to provide novelcompositions containing EBV transformed human B-cells obtained from SCIDmice which predominantly secrete human IgG's.

[0047] It is a more specific object of the invention to provide novelcompositions containing EBV transformed human B-cells whichpredominantly secrete human IgG's produced by a method comprising:

[0048] (i) antigen priming of naive human splenocytes in vitro;

[0049] (ii) transferral of resulting in vitro antigen primed naivesplenocytes to an immunocompromised animal donor, e.g., a SCID mouse;

[0050] (iii) boosting the immunocompromised animal donor, e.g., SCIDmouse, with antigen;

[0051] (iv) isolation of human antibody producing B-cells from theantigen boosted immunocompromised donor, e.g., SCID mouse; and

[0052] (v) EBV transformation of said isolated human antibody producingB-cells.

[0053] It is another specific object of the invention to produce RSVneutralizing human monoclonal antibodies having an affinity to the RSVF-protein of ≦2×10⁻⁹ Molar.

[0054] It is still another object of the invention to provide EBVimmortalized cell lines which secrete RSV neutralizing human IgGmonoclonal antibodies having an affinity to the RSV F antigen of ≦2×10⁹Molar.

[0055] It is a more specific object of the present invention to providetwo EBV immortalized cell lines, RF-2 and RF-1, which respectivelysecrete human monoclonal antibodies also referred to as RF-2 and RF-1which neutralize RSV in vivo and each possess an affinity for the RSVF-protein of ≦2×10⁻⁹.

[0056] It is another object of the invention to transfect eukaryoticcells with DNA sequences encoding the RF-1 or RF-2 heavy and lightvariable domains to produce transfectants which secrete human antibodiescontaining the variable domain of RF-1 or RF-2.

[0057] It is a more specific object of the invention to providetransfected CHO cells which express the RF-1 or RF-2 heavy and lightvariable domains.

[0058] It is another object of the invention to treat or prevent RSVinfection in humans by administering a therapeutically orprophylactically effective amount of RSV neutralizing human monoclonalantibodies which are specific to the RSV F-protein and which exhibit aKd for the RSV F-protein of ≦2×10⁻⁹ molar.

[0059] It is a more specific object of the invention to treat or preventRSV infection in humans by administering a therapeutically orprophylactically effective amount of RF-1 or RF-2 or a human monoclonalantibody expressed in a transfected eukaryotic cell which contains andexpresses the variable heavy and light domains of RF-1 or RF-2.

[0060] It is another object of the invention to provide vaccines fortreating or preventing RSV infection which comprise a therapeutically orprophylactically effective amount of human monoclonal antibodiesspecific to the RSV F-protein having a Kd for the RSV F-protein of≦2×10⁻⁹ molar, which neutralize RSV in vitro, in combination with apharmaceutically acceptable carrier or excipient.

[0061] It is a more specific object of the invention to provide vaccinesfor treating or preventing RSV infection which comprise atherapeutically or prophylactically effective amount of RF-1 or RF-2 orhuman monoclonal antibodies derived from a transfected eukaryotic cellwhich contains and expresses DNA sequences encoding the variable heavyand light domains of RF-1 or RF-2, in combination with apharmaceutically acceptable carrier or excipient.

[0062] It is another object of the present invention to provide a methodfor diagnosis of RSV infection by assaying the presence of RSV inanalytes, e.g., respiratory fluids using human monoclonal antibodieswhich possess an affinity for the RSV fusion (F) protein or ≦2×10⁻⁹molar.

[0063] It is still another object of the invention to provide novelimmunoprobes and test kits for detection of RSV infection which comprisehuman monoclonal antibodies specific to the RSV F-protein, which possessan affinity for the RSV F protein of ≦2×10⁻⁹ molar, which antibodies aredirectly or indirectly attached to a suitable reporter molecule, e.g.,an enzyme or a radionuclide. In the preferred embodiment these humanmonoclonal antibodies will comprise RF-1 or RF-2 or recombinant humanmonoclonal antibodies produced in eukaryotic cells, e.g., CHO cells,which are transfected with the variable heavy and light domains of RF-1or RF-2.

BRIEF DESCRIPTION OF THE INVENTION

[0064] The present invention in its broadest embodiments relates tonovel methods for making human antibodies to desired antigens,preferably antigens involved in prophylaxis, treatment or detection of ahuman disease condition. These methods comprise antigen priming ofnative human splenocytes in vitro, transferral of the resultant in vitroantigen primed splenocyte cells to an immunocompromised donor, e.g., aSCID mouse, and boosting said immunocompromised donor with antigen.

[0065] The present invention also relates to methods for producingEpstein-Barr Virus (EBV) immortalized B-cells which favors theproduction of cells which secrete IgGs comprising: antigen priming ofnaive human splenocytes in vitro; transferral of resultant in vitroantigen primed splenocytes to an immunocompromised donor, e.g., a SCIDmouse; boosting the immunocompromised donor with antigen; isolatinghuman antibody secreting B-cells, preferably IgG secreting, from theantigen boosted immunocompromised donor, e.g., SCID mouse; and EBVtransformation of said isolated human antibody secreting cells.

[0066] The present invention more specifically relates to improvedmethods for making human antibodies to RSV, in particular the RSV fusion(F) protein which exhibit high affinity to RSV F-protein and which alsoneutralize RSV infection, as well as the human monoclonal antibodieswhich result from these methods. This is preferably effected by primingof naive human splenocytes in vitro with Il-2 and optionally the RSVF-protein; transferral of the resultant in vitro primed splenocyte cellsto an immunocompromised donor, e.g., a SCID mouse, and boosting with RSVF-protein to produce human B-cells which secrete neutralizing anti-RSVF-protein human antibodies having high affinity to the RSV F-protein,i.e., ≦2×10⁻⁹ molar.

[0067] The resultant B-cells are preferably immortalized so as toprovide a constant stable supply of human anti-RSV F-protein monoclonalantibodies. In the preferred embodiment B-cells are isolated from theantigen boosted SCID mouse and transformed with EBV virus to produce EBVtransformed human B-cells which predominantly secrete human IgGs.

[0068] These cells are then cloned to select EBV transformed cell lineswhich secrete human monoclonal antibodies having high affinity to RSVF-protein, i.e. ≦10⁻⁷ and preferably ≦2×10⁻⁹ molar.

[0069] The present invention also relates to the use of such anti-RSVF-protein human monoclonal antibodies as therapeutic and/orprophylactic, as well as diagnostic agents. As noted, the subjectmethods result in the generation of human monoclonal antibodies whichexhibit high affinity to the RSV F-protein, i.e., which possess a Kd forthe RSV F-protein of ≦2×10⁻⁹ molar, which also neutralize RSV in vitro.Therefore, these antibodies are ideally suited as prophylactic andtherapeutic agents for preventing or treating RSV infection given thefact that the RSV F-protein is a surface protein which is highlyconserved across different RSV isolates. Also, given the high affinityand specificity of the subject human monoclonal antibodies to RSVF-protein, they also may be used to diagnose RSV infection.

[0070] More specifically, the present invention provides two particularhuman monoclonal antibodies to the RSV F-protein, i.e., RF-1 and RF-2,as well as recombinant human antibodies derived therefrom, which arepreferably produced in CHO cells, which cells have been transfected withDNA sequences encoding the variable heavy and light domains of RF-1 orRF-2. These antibodies are particularly useful as prophylactic and/ortherapeutic agents for treatment or prevention of RSV infection.Moreover, these antibodies are useful as diagnostic agents because theybind the RSV F-protein with high affinity, i.e., each possess affinityfor the RSV F-protein of ≦2×10⁻⁹. They are especially useful astherapeutic agents because of their high affinity and specificity forthe RSV F-protein, and their ability to effectively neutralize RSVinfection in vitro.

BRIEF DESCRIPTION OF THE FIGURES

[0071]FIG. 1 depicts immunoblot of F protein with anti-F proteinhu-SPL-SCID sera: Notice (A) and denatured (B) F protein was run inSDS-PAGE and transferred to nitrocellulose by Western blot.Nitrocellulose strips were reacted with positive control mouse anti-Fprotein MAb (lanes 1A and 1B), negative control hu-SPL-SCID serumanti-TT (lanes 2A and 2B) and hu-SPL-SCID anti-F protein sera from mice#6 (lanes 3A and 3B), #3 (lanes 4A and 4B) and #4 (lanes #5A and 5B).

[0072]FIG. 2 depicts immunofluorescence of HEP-2 cells with hu-SPL-SCIDsera anti-F protein. Uninfected (left) and RSV-infected HEp-2 cells werereacted with serum from hu-SPL-SCID mouse #6 diluted 1:50 taken 15 daysafter boost. Binding was revealed GAH IgG-FITC.

[0073]FIG. 3 depicts the reactivity of purified RF-1 and RF-2 to plasticbound affinity purified RSV F-protein. The reactivity of a referencehuman anti-RSV serum, LN, is also recorded. The ELISA plate was coatedwith 50 ng RSV F-protein.

[0074]FIG. 4 depicts IEF of RF-1 (lane 2) and RF-2 (lane 3) human MAbpurified from tumor cell supernatants. IEF was performed on a pHgradient of 3-10. Lane 1 represents the pi standards.

[0075]FIG. 5 depicts indirect Immunofluorescence flow cytometry assay ofHEp-2 cells and HEp-2-cells infected with RSV, 1×10⁶, incubated withvarious amounts of RF-1 and subsequently with a FITC-labeled GAH IgG.The relative average intensity of the entire population is recorded.

[0076]FIG. 6 depicts NEOSPLA vector used for expression of humanantibodies. CMV=cytomegalovirus promoter. BETA=mouse beta globin majorpromoter. BGH=bovine growth hormone polyadenylation signal. SVO=SV40origin of replication. N1=Neomycin phosphotransferase exon 1.N2=Neomycin phosphotransferase exon 2. LIGHT=Human immunoglobulin kappaconstant region. Heavy=Human immunoglobulin gamma 1 or gamma 4 PEconstant region. L=leader. SV=SV40 polyadenylation region.

[0077]FIG. 7a depicts the amino acid and nucleic acid sequence of thevariable light domain of RF-1.

[0078]FIG. 7b depicts the amino acid and nucleic acid sequence of thevariable heavy domain of RF-1.

[0079]FIG. 8a depicts the amino acid and nucleic acid sequence of thevariable light domain of RF-2.

[0080]FIG. 8b depicts the amino acid and nucleic acid sequence of thevariable heavy domain of RF-2.

[0081]FIG. 9a depicts the amino acid and nucleic acid sequence of theRF-1 light chain, the leader sequence, and the human kappa constantdomain sequence.

[0082]FIG. 9b depicts the amino acid and nucleic acid sequence of theRF-1 heavy chain, a leader sequence, and the human gamma/constant domainsequence.

[0083]FIG. 9a depicts the human constant domain sequence.

[0084]FIG. 10 depicts schematically the NEOSPLA vector, referred to asNSKE1 containing the RF-1 nucleic acid sequence and human gamma/constantdomain set forth in FIGS. 9a-9 c.

[0085]FIG. 11a depicts the amino acid and nucleic acid sequence of theRF-2 light chain, leader sequence, and human Kappa constant domain.

[0086]FIG. 11b depicts the amino acid and nucleic acid sequence of theRF-2 heavy chain, leader sequence, and human gamma/constant domain.

[0087]FIG. 11c depicts the amino acid and nucleic acid sequence of thehuman gamma/constant domain.

[0088]FIG. 12 depicts schematically the NEOSPLA expression vector,referred to as NSKG1 containing the RF-2 nucleic acid sequences andhuman gamma/constant domain sequences set forth in FIGS. 11a-11 c.

DETAILED DESCRIPTION OF THE INVENTION

[0089] As discussed, the present invention provides a novel highlyefficient method for producing human monoclonal antibodies to desiredantigens, preferably antigens which are involved in a human diseasecondition. Antigens involved in a human disease condition typically willbe surface antigens which comprise suitable therapeutic targets forantibodies. For example, this includes surface proteins of viruses andantigens expressed on the surface of human cancer cells. In thepreferred embodiment, the surface antigen will comprise the fusionprotein (F-protein) of RSV.

[0090] Human disease conditions includes by way of example viralinfections, e.g., RSV, papillomavirus, hepatitis, AIDS, etc., cancer,bacterial infections, yeast infections, parasite infection, e.g,malaria, etc. Essentially, human disease conditions are intended toembrace any human disease condition potentially preventable or treatableby the administration of human monoclonal antibodies specific to aparticular antigen.

[0091] The subject method for producing human monoclonal antibodiesessentially involves the combination of in vitro priming of naive humanspleen cells, transferral of these spleen cells to immunocompromiseddonors, i.e., SCID mice, followed by antigen boosting of SCID mice whichhave been administered said spleen cells. It has been surprisinglydiscovered that the combination of these two known methods for producinghuman antibodies results in synergistic results. Specifically, itresults in very enhanced antigen specific responses to the immunizingantigen as well as very high titers of human monoclonal antibodies ofthe IgG isotype. More specifically, it has been found that thiscombination results in unprecedented high secondary responses: the humanIgG responses in the hu-SPL-SCID serum were 10-fold higher than thoseresulting from transfer of naive cells in SCID and specific antibodyresponses were 1000-fold increased. Also, the resulting antibodies arefound to be of high affinity and specificity comparable to antibodiesproduced in experimentally hyperactive immune animals. It has also beenfound that when using naive spleen cells, to obtain such unexpectedresults it is necessary to challenge with antigen both in vitro andafter introduction into the resultant hu-SPL-SCID mouse. Also, it ispreferable but not essential to introduce additional fresh non-primedspleen cells to the hu-SPL-SCID donor just prior to antigen boosting.This has been found to result in still further enhancement of theantibody response.

[0092] The present invention was developed after an optimal in vitroprimary and boosting protocol for the generation of secondary responsesfrom naive human spleen cells had previously been disclosed Brams et al,Hum. Antibod. Hybridomas (1993), 4, 57-65. The protocol Brams et al,Hum. Antibod. Hybridomas (1993), 4, 57-65 was found to provide forantigen specific IgG responses about 2 to 10 times higher than obtainedfrom cultures subjected to one antigen challenge. This in vitroimmunization (IVI) protocol was developed and optimized using verydifferent antigens, i.e., horse ferritin (HoF), calmodulin, prostatespecific antigen (PSA), mouse IgG, transferrin, Keyhole LimpetHemocyanine (KLH) di-nitro phenyl (DNP) bound to T-cell dependentprotein carriers and RSV fusion (F) protein.

[0093] Essentially, this protocol involves restimulation of the spleencell culture on day 1 after culturing is started with antigen togetherwith autologous spleen cells in a 1:1 ratio. It has been demonstratedthat the IgG responses measured using this protocol were the result ofrepeated antigen exposure, and are equivalent to secondary responses.

[0094] These experiments further demonstrated that intact spleens werethe optimal source of lymphocytes, including trauma- and ITP spleens. Bycontrast, peripheral blood lymphocytes (PBLs), and cells from tonsils orlymph nodes proved to be inferior for induction of antigen-specificresponses. Moreover, depletion or neutralization of any cellularcomponent resulted in inferior responses Boerner et al. J. Immunol.(1991), 147:86-95. Also, these experiments indicated that for a givenspleen cell preparation and antigen, that there exists a unique optimalantigen concentration.

[0095] Therefore, having established an optimal in vitro primary andboosting protocol for generation of secondary responses from naive humanspleen cells; it was conceived to test this protocol in combination withprevious in vivo methods for producing human monoclonal antibodies,i.e., the SCID mouse. It was unknown prior to testing what effect, ifany, administration of antigen primed spleen cells would have on theresultant production of human monoclonal antibodies to a given antigenby the SCID mouse or the ability of human lymphocytes to be maintainedtherein. However, it was hoped that this would provide for enhancedantigen boost and enhanced expression of the in vitro antigen primednaive spleen cells.

[0096] In this regard, it has been previously reported that humanlymphocytes can establish themselves and remain alive for several monthsin SCIDs McCune et al, Science (1988), 241, 1632-1639, Lubin et al,Science (1991), 252:427. However, as noted, surpa previous methods usingSCIDs or human monoclonal antibodies to antigens have used cells fromdonors previously exposed to the antigen either naturally or byvaccination and have typically not resulted in high human antibodytiters.

[0097] Quite surprisingly, it was found that combination of in vitroprimary and boosting protocol for generation of secondary responses fromhuman naive spleen cells Brams et al. Hum. Antibod. Hybridomas (1993),4, 57-65 in the hu-SCID model resulted in synergistic results asevidenced by highly significant antigen specific IgG responses to theimmunizing antigen.

[0098] Further, it was also discovered that the combination of thesemethods (using horse ferritin (HoF) as a model antigen) that:

[0099] (i) introduction of an in vitro immunization step prior totransfer into SCIDs is essential for reliably inducing significantantigen-specific responses;

[0100] (ii) human cells must be transferred into the peritoneum toachieve optional maintenance of human splenocytes in the SCID mouse;

[0101] (iii) optimal in vitro cultivation is about three days;

[0102] (iv) use of IL-2 and optionally IL-4 or IL-6 in vitro results inhighest antibody titers of antigen specific responses in the hu-SPL-SCIDmice;

[0103] (v) the hu-SPL-SCID in mouse is preferably boosted with antigenemulsified in an adjuvant, e.g., Freunds Complete Adjuvant (FCA) and/orAlum;

[0104] (vi) killing or neutralization of NK cells, whether of murine orhuman origin surprisingly has no benefit on antibody production.However, it was found that use of the SCID-beige mouse, an NK low line,as the host for the in vitro primed cells, provides for a superiorresponse when boosting is effected using a combination of adjuvants,i.e., FCA and Alum.

[0105] (vii) spleens, but not lymph nodes of ⅓ of the hu-SPL-SCID micewere enlarged up to 25 times compared to normal SCIDs. Moreover, ofthese up to two-thirds of the cells in such spleens tested positive fornormal human lymphocyte membrane markers.

[0106] More specifically, the subject method comprises priming naivehuman splenocytes in vitro, for about 1 to 10 days, preferably about 3days with antigen, transferral of the primed cells to a SCID mouse, andsubsequently boosting the mouse with antigen about 3 to 14 days later,preferably about 7 days later. This has been demonstrated to result inhigh antigen specific IgG responses in the sera of the resultanthu-SPL-SCID mouse from about day 24 onwards. Typically, the serumend-dilution titers are about 10⁶ (half maximal responses atapproximately 50 mg IgG/ml) using a naive antigen, horse ferritin and10⁷ (half maximal responses at approximately 5 mg IgG/ml) when a recallresponse is induced with a viral antigen, i.e., the fusion protein ofRSV. It is expected based on these results that similar responses willbe obtained using other antigens.

[0107] As noted, optimal induction of the desired antibody responserequires antigen challenge of the human cells both in vitro and in vivoin the hu-SPL-SCID mouse. It was also found that IL-2 is necessaryduring in vitro priming, and that IL-4 and IL-6 administeredconcomitantly with IL-2 further enhanced responses in the hu-SPL-SCIDmouse. Moreover, SCID reconstitution is facilitated but was notdependent on concomitant intraperitoneal administration of irradiatedallogeneic lymphocytes.

[0108] It was further discovered that there was significant variation inthe antibody responses from one spleen to another. For example, somespleens required concomitant administration of antigen and freshautologous spleen cells on day 10 for generation of antigen specificantibody responses. Also, it was found that the level of antibodyresponses varied somewhat in different hu-SPL-SCID mice. However, basedon the teachings in this application, one skilled in the art can readilyselect suitable conditions so as to produce an optimal antigen specificantibody response to a given antigen.

[0109] For example, by testing several different spleen preparations fortheir ability to produce specific antibody in culture, e.g., after tendays of in vitro immunization, one can identify the highest responder.Moreover, since large numbers of cells are prepared and frozen from eachspleen, it is possible to set up a new in vitro immunization for threedays from the selected spleen and follow up with transfer in SCID mice.By contrast, other cellular materials, e.g., peripheral blood cells arenot amenable to such optimization, given the fairly limited amount ofPBL's recoverable from one donor in a single transferral.

[0110] As previously noted, in contrast to previous reports, it wasfound that for the present method, when peripheral blood cells wereused, neutralization of human NK activity had no effect on spleens.Moreover, neutralization of SCID NK cells with complement fixinganti-asialo GMI antibodies decreased antigen-specific IgG responses. Bycontrast, use of the SCID/beige mouse, a strain with reduced NK celllevels did provide for significantly increased antigen specific IgGresponses compared to normal SCID.

[0111] Additionally, two immunization routes, intravenous (IV) andintraperitoneal (IP) were compared for their ability to provide forreconstitution of SCID mice, i.e., maintenance of spleen cells thereinand the production of human antibodies. It was found that the peritoneumwas the optimal site of cell transfer and immunization. Moreover, date,transfer of cells intravenously has never been found to result inrepopulation when more than 0.01 μg/ml human IgG was detected in themouse serum.

[0112] It was also found that the resultant IgG concentrations directlycorrelated with the number of transferred human cells. For example,repopulation of SCIDs was 92% when 5×10⁶ in vitro primed spleen cellswere injected intraperitoneally, and virtually 100% when 5×10⁷ in vitroprimed spleen cells were injected intraperitoneally. One skilled in theart can, based on the teachings in this application, select an optimalnumber of injected in vitro primed spleen cells. In general, this willrange from about 10⁴ to about 10⁸ cells, more preferably about 10⁶ to10⁸ cells, and most preferably at least about 10⁷ to 10⁸ cells.

[0113] It was also found that the antibody response is affected by thepresence of the particular adjuvant. More specifically, it was observedthat maximal human antibody responses were achieved when the hu-SPL-SCIDmice were boosted with antigen emulsified in Complete Freund's adjuvant(CFA) or using CFA and Alum together. Tests in hu-SPC-SCID boosted withferritin showed that CFA was a better adjuvant than Alum, eliciting 33mg and 13 mg/ml human IgG respectively. Combination of CFA and Alum didnot improve response in SCID. However, use of these adjuvants inSCID/beige-hu (which mice comprise a mutation resulting in reduced NKcell activity) results in 8-10 fold increase in IgG production comparedto CFA alone. However, it is expected that other adjuvants, orcombinations thereof, may also produce similar or even enhanced results.The highest total human IgG concentrate using Complete Freund's adjuvantand Alum together was about 10 mg/ml, and the specific highest IgGconcentration was about 500 μg/ml monoclonal antibody equivalent.

[0114] Using this method with ferritin produced polyclonal antibodyresponses comparable to that obtained in hyperimmune goats, rabbits andpigs in terms of specificity, reactivity, and use of Ig chain isotypes.The hu-SPL-SCID serum antibodies were mostly IgG, bound only to cellsfrom tissues high in ferritin, and not to cells from ferritin-low orferritin-negative tissues, and recognized both natural ferritin as wellas denatured ferritin in a Western blot. These results are extremelyunexpected both in antibody concentration and the antigen specificity ofhuman antibodies obtained. Moreover, similar results are obtained usingdifferent antigens.

[0115] After injection, it is found that human cells tend to accumulateat two sites, i.e., the peritoneum and the mouse spleen. While no morethan about 7% of human cells were found in the blood, the lymph nodesand the liver were of human antigen, between 25% and 33% of the cellswere of human origin in enlarged spleens and in the occasional tumors insome animals. These human cells were almost exclusively B and T-cells,with a small amount of CD14⁺ cells, mostly monocytes, in the enlargedspleens.

[0116] These results were determined by flow cytometry investigatingspleen, lymph nodes, liver and peritoneum. In those cases that the humansplenocytes repopulated the spleen, it was found that the spleens wereoften enlarged, up to 25 times the size of native SCID spleens. Thehuman cells constituted up to about 30% of the total number of cells inthe spleen when measured immediately after extraction, with theremainder of unknown origin. However, after 3 days in culture, amajority of surviving cells were found to be of human origin as thecells bound antibodies and exhibited no cross reactivity with mouselymphocytes.

[0117] It was further observed that the reconstituted mice could bedivided into two groups, those with normal size spleens and those withenlarged spleens. Hu-SPL-SCID mice with enlarged spleens, i.e., 25 timesnormal size had human IgG levels approximately 150 times higher thanthose with normal spleens, and the level of antigen specific human IgGwas approximately 10,000 higher in those with normal size spleens whichwere treated similarly. It was also found that the relative affinity ofthe antigen specific response increased throughout the response,indicating that a higher percentage of the total immunoglobulin pool wascomprised of antibodies having better binding properties. These resultsindicate that the system is antigen driven.

[0118] These results are highly significant and indicate that it shouldgenerally be possible to rescue human cells from the hu-SPL-SCID and usesame for generating combinatorial human antibody gene libraries therebyresulting in human monoclonal antibodies of high affinity andspecificity that may be used clinically and/or diagnostically.

[0119] More specifically, the present invention provides novel humanmonoclonal antibodies to the RSV F-protein which exhibit high affinityto the RSV F-protein, i.e., ≦2×10⁻⁹ molar protein and which humanmonoclonal antibodies are capable of neutralizing RSV in vitro. Thepresent invention further provides methods for manufacture of such humanmonoclonal antibodies to the RSV F-protein.

[0120] In general, such human antibodies are produced by in vitroimmunization of naive human splenocytes with RSV F-protein, transferralof such in vitro immunized human splenocytes into an immunocompromisedanimal donor, i.e., a SCID mouse, boosting said animal with RSVF-protein, and isolation of human B cells therefrom which secrete humanmonoclonal antibodies to the RSV F-protein, immunization of said human Bcells, and cloning of said immunilized B cells to select cells whichsecrete human monoclonal antibodies having a high affinity to RSVF-protein, preferably at least 10⁻⁷ molar and more preferably ≦2×10⁻⁹molar.

[0121] As discussed, it has been discovered that the combination of invitro immunization, in particular of human splenocytes, i.e., which haveor have not been previously exposed to the RSV F-antigen and transferredto an immunocompromised animal donor, i.e., SCID mouse which is thenboosted with RSV F-protein antigen affords significant advantagesrelative to conventional methods for making human antibodies in SCIDmice. Namely, it provides for very high antibody titers, i.e., thehighest anti-F protein titers being about 10⁻⁷, high IgG concentrations,i.e., about 3 mg/ml for the highest responders. Moreover, this methodallows for the production of human antibodies having highly advantageouscombinations of properties, i.e., which exhibit both high affinity tothe RSV F-protein and which moreover display substantial in vitroneutralizing activity.

[0122] As described in greater detail in the examples, the presentinventors have isolated two human monoclonal antibodies, RF-1 whichexhibits an affinity constant Ka to the F-protein, Ka=10¹⁰ M whendetermined by plasmon resonance, and RF-2 which exhibits an affinityconstant of Ka=5×10⁸ M when determined by titration microcolorimetry.Also, the calculated Kd of RF-2 was 2×10⁻⁹ M. Moreover, both of theseantibodies display in vitro virus neutralizing properties atconcentrations of between 8 and 120 ng/ml as well as exhibiting anability to inhibit the fusion of previously RSV infected cells.Significantly, this in vitro neutralization activity is applicableagainst a broad variety of different wild and laboratory RSV strains,both of the A and B virus types.

[0123] Given these results, i.e. the high affinity of the subjectantibodies to the RSV F-protein, which comprises a surface proteinexpressed on the surface of RSV infected cells, as well as ability toeffectively neutralize the virus, and to inhibit fusion of virallyinfected cells, the subject human monoclonal antibodies should besuitable both as therapeutic and prophylactic agents, i.e., for treatingor preventing RSV infection in susceptible or RSV infected subjects. Asnoted, RSV infection is particularly prevalent in infants, as well as inimmunocompromised persons. Therefore, the subject monoclonal antibodieswill be particularly desirable for preventing or treating RSV infectionin such subjects.

[0124] Moreover, given the human origin of the subject monoclonalantibodies, they are particularly suitable for passive immunotherapy.This is because they likely will not be subject to the potentialconstraints of murine monoclonal antibodies, i.e., HAMA responses andabsence of normal human effector functions. In fact, based on thecharacterization of the subject human monoclonal antibodies (describedin examples infra), it would appear that both RF-1 and RF-2 exhibitsubstantially greater in vitro neutralization activity and ability toinhibit fusion of previously infected RSV cells than previouslydisclosed murine or chimeric anti-F protein antibodies and human Fabfragments derived from recombinational libraries. Also, given theirhuman origin it is expected that such neutralization activity will bemaintained upon in vivo administration.

[0125] Another advantage of the subject human monoclonal antibodies istheir substantial absence of reactivity with normal tissues. As showninfra, the subject human monoclonal antibodies bind only to RSV infectedcells, not to cell lines representing lymphoid tissue, liver, prostrateor laryngeal epidermis. Therefore, these antibodies upon in vivoadministration should efficiently bind to RSV infected cells and not tonormal tissues and thereby should provide for neutralization of RSVinfection. Further, based on the disclosed properties, it is expectedthat the subject human monoclonal antibodies to the RSV F-protein may beused to protect susceptible hosts against RSV infection.

[0126] More specifically, the subject human monoclonal antibodies to theRSV F-protein are produced by obtaining human splenocytes, e.g., from atrauma or ITP source, which are then primed in vitro. This essentiallycomprises culturing said naive human splenocytes in vitro in thepresence of a sufficient amount of Il-2 and optionally RSV F-protein toinduce immunization, also referred to as antigen priming. In general,the amount of RSV F-protein that may be used ranges from about 1 to 200ng/ml RSV protein, more preferably 10 to 100 ng/ml, and most preferablyabout 40 ng/ml of RSV F-protein.

[0127] The in vitro culture medium will preferably also containlymphokines, in particular IL-2 and optionally IL-4 and IL-6. The amountthereof will be amounts which provide for immunization and the desiredproduction of antibody producing cells. For example, in the case ofIL-2, an amount ranging from about 5 to 200 IU/ml, and more preferablyfrom about 10 to 50 IU/ml, most preferably 25 IU/ml is suitable.

[0128] This culture medium will also contain other constituentsnecessary to maintain the viability of human splenocytes in culture,e.g., amino acids and serum. In the examples, a culture mediumcontaining IMDM supplemented with 2 mM glutamine, 2 mM sodium pyruvate,non-essential amino acids, 25 IU/ml IL-2 and 20% fetal calf serum wasused. However, one skilled in the art, based on the teachings in thisapplication, can vary the culture medium using routine optimization.

[0129] The in vitro immunization step will be effected for a timesufficient to induce immunization. In general, the cells will becultured in the presence of RSV F-protein from about 1 to 10 days andpreferably for about 3 days. However, this will vary dependent, e.g.,upon the particular spleen sample. Similarly, one skilled in the art,based on the teachings in this application and using known methods maydetermine a suitable duration for the in vitro immunization step.

[0130] The antigen used for the in vitro immunization will preferably bea purified RSV F-protein so as to ensure that the splenocytes areimmunized against the F-protein and not against other useless(non-surface) antigens. Methods for obtaining purified RSV protein areknown in the art. The present inventors in particular utilized themethod of Walsh et al. J. Gen Virol., 70, 2953-2961, 1989. However, theparticular method is not critical provided that RSV F-protein ofsufficient purity to obtain human monoclonal antibodies havingspecificity to the RSV F-protein are obtained. Alternatively, the RSVF-protein may be produced by recombinant methods as described in U.S.Pat. No. 5,288,630 issued on Feb. 22, 1994.

[0131] After in vitro immunization, the RSV F-protein immunized orprimed naive human splenocytes are then introduced into animmunocompromised donor, i.e., a SCID mouse. This is preferably effectedby intraperitoneally administering the RSV F-protein primed humansplenocytes into SCID mice. The number of such splenocytes which isadministered will typically vary from about 10⁴ to 10⁸ spleen cells,with about 10⁷ to 10⁸ spleen cells being preferred. The number of suchcells is that which results in the desired reconstitution, i.e., SCIDmice which produce recoverable concentrations of human antibodiesspecific to the RSV F-protein. Preferably, such spleen cells will besuspended in HBSS at a concentration of about 8×10⁸ cells/ml prior toadministration.

[0132] After intraperitoneal transferral of splenocytes, the SCID miceare then boosted with the RSV F-protein. This is effected at a timesufficiently proximate to the transferal of splenocytes such that thedesired production of human anti-RSV F-protein antibodies is realized.In general, this may be effected 3 to 14 days after transferral, andoptimally about 7 days after transferral. Preferably, said antigenadministration will be effected intraperitoneally. The amount of RSVF-protein administered will range from about 1 to 50 μg and preferablyabout 1 to 10 μg. In the examples, 5 μg protein was administered.However, the amount and time of immunization may vary dependent upon theparticular mouse, spleen sample, and purity of RSV F-protein.

[0133] Preferably, antigen boosting will be effected in the presence ofan adjuvant, e.g., Complete Freund's Adjuvant, Alum, Saponin, etc., withComplete Freund's Adjuvant (CFA) and Alum being preferred. However, itis expected that other known adjuvants may be substituted to obtainsubstantially equivalent or even enhanced results.

[0134] After antigen boosting, the SCID mice are then bled, e.g., tailbled, and their serum tested for human IgG concentration and anti-Fprotein antibody titers. Those animals which exhibit the highestantibody titers and concentration are then used for recovery of humanIgG secreting cells.

[0135] It has been discovered that SCID mice having the highest anti-Fhuman antibody titers developed large abdominal tumors which provide agood source of human antibody secreting cells. Preferably, these tumorsare recovered by excision under sterile conditions, single cellsuspensions are prepared, and the cells are then washed and cultured. Inthe examples, the cells are washed with IMDM containing 2% fetal calfserum, and the cells cultured in suspension of 10⁶ cells/ml in T-25flasks containing IMDM with 10% FCS. However, such culturing conditionsmay be varied by one skilled in the art.

[0136] These cells are then immortalized preferably using EBV.Immortalized cells which secrete anti-F protein antibodies are thenidentified by known methods, e.g., ELISA. As noted, this method has beendemonstrated to result in the identification of two distinct humanmonoclonal antibodies which specifically bind RSV F-protein, i.e. RF-1and RF-2. However, based on the teachings in their application, inparticular the examples, other human monoclonal antibodies to the RSVF-protein having similar properties may be obtained by one skilled inthe art absent undue experimentation. These antibodies are distinctgiven the fact that most were generated in two different experiments,using different SCID mice. The cell lines which express RF-1 and RF-2have been maintained in culture for prolonged time, i.e., about 18 and16 months respectively; dividing with an approximate doubling time ofabout 36-48 hours. The specific antibody concentration is on averageabout 0.8-1 μg/ml in a culture seeded at 0.5×10⁶ cells/ml grown forthree days.

[0137] As discussed in greater detail infra, both RF-1 and RF-2 are IgG(1, k) with half-maximal binding to F-protein in ELISA at 0.6 and 1ng/ml respectively, and exhibiting isoelectric points of about 8.8 and8.9 respectively.

[0138] Moreover, these antibodies exhibit high affinity to the RSVF-protein. Specifically, for RF-1 the dissociation constant for RF-1 asdetermined by plasmon resonance on an IASYS machine is about 10⁻¹⁰ M.The Ka constant for RF-2 is similarly high; when determined by titrationmicrocolorimetry according to Wiseman et al. (1989) and Robert et al.(1989) it is about 2×10⁻⁹ M.

[0139] Additionally, these antibodies have been demonstrated toeffectively bind RSV infected cells, while not binding normal humancells tested, e.g., respiratory tract lining (HEp-2, a laryngealepidermoid carcinoma, CCL 23), liver (HepG2, a human hepatoma cell line,HB 8068), lymphoid tissue, SB, a human B lymphoblastoid cell line, cat.no. CCL 120 and HSB, a T lymphoblastoid line, cat. no. CCL 120.1, andprostrate (LNCaP.FGC, a human prostrate adenocarcinoma line, cat. no.CRL 1740).

[0140] Significantly, both RF-1 and RF-2 both have been shown to exhibitsubstantial in vitro RSV viral neutralization. This was demonstrated intwo different assays (described in greater detail infra), i.e., aninfection neutralization assay effected by pre-reacting the virus withpurified monoclonal antibody prior to its addition to cells (whichmeasures ability of antibody to inhibit virus infectivity) and a fusioninhibition assay which measures the ability of the monoclonal antibodyto inhibit virus growth and expansion after virus entry into the cell.

[0141] Moreover, as discussed in greater detail infra, both RF-1 andRF-2 inhibited virus infection of twelve different isolates atconcentrations respectively ranging from about 30 ng/ml to 1000 ng/ml.Thus, RF-2 apparently performs better than RF-1, yielding to 50% virusinhibition (ED50) at concentrations which are about 1.25 to 10 timeslower than RF-1.

[0142] By contrast, higher concentrations of monoclonal antibody arerequired to inhibit fusion and viral antigen expression in previouslyinfected cells, with RF-1 being about 5 to 10 times more potent thanRF-2. Moreover, both RF-1 and RF-2 were effective against a Type B RSV,Type B prototype RS 6556, and a Type A RSV, Type A prototype RS Long.Thus, the in vitro results indicate that the subject human monoclonalantibodies may be used to treat or prevent RSV infection caused bydifferent RSV strains, both of Type A and Type B prototype. As discussedpreviously, the RSV F-protein is fairly well conserved in different RSVisolates. Therefore, it is likely that the subject monoclonal antibodiesto the RSV F-protein bind to a conserved epitope of RSV F-protein.

[0143] As discussed, the subject human monoclonal antibodies orrecombinant human antibodies containing the variable heavy and lightsequences therefrom (preparation discussed infra) will be used astherapeutic and prophylactic agents to treat or prevent RSV infection bypassive antibody therapy. In particular, the DNA sequence encoding theseDNA variable domains may be incorporated in IDEC's proprietaryexpression vector which is depicted in FIG. 2. This version issubstantially described in commonly assigned U.S. Ser. No. 08/379,072,filed on Jan. 25, 1995, herein incorporated by reference. This vectorconstant human constant domain, for example, human gamma 1, human gamma4 or a mutated form thereof referred to as gamma 4 PE. (See U.S. Ser.No. 08/379,072, incorporated by reference herein.) In general, this willcomprise administering a therapeutically or prophylactically effectiveamount of the subject human monoclonal antibodies to a susceptiblesubject or one exhibiting RSV infection. A dosage effective amount willpreferably range from about 50 to 20,000 μg/Kg, more preferably fromabout 100 to 5000 μg/Kg. However, suitable dosages will vary dependenton factors such as the condition of the treated host, weight, etc.Suitable effective dosages may be determined by those skilled in theart.

[0144] The subject human monoclonal antibodies may be administered byany mode of administration suitable for administering antibodies.Typically, the subject antibodies will be administered by injection,e.g., intravenous, intramuscular, or intraperitoneal injection, or morepreferably by aerosol. As previously noted, aerosol administration isparticularly preferred if the subjects treated comprise newborn infants.

[0145] Formulation of antibodies in pharmaceutically acceptable form maybe effected by known methods, using known pharmaceutical carriers andexcipients. Suitable carriers and excipients include by way of examplebuffered saline, bovine serum albumin, etc.

[0146] Moreover, the subject antibodies, given their high specificityand affinity to RSV infected cells possess utility as immunoprobes fordiagnosis of RSV infection. This will generally comprise taking asample, e.g. respiratory fluid, of a person suspected of having RSVinfection and incubating the sample with the subject human monoclonalantibodies to detect the presence of RSV infected cells.

[0147] This will involve directly or indirectly labeling the subjecthuman antibodies with a reporter molecule which provides for detectionof human monoclonal antibody—RSV immune complexes. Examples of knownlabels include by way of example enzymes, e.g. β-lactamase, luciferase,etc. and radiolabels.

[0148] Methods for effecting immunodetection of antigens usingmonoclonal antibodies are well known in the art. Also, the subjectanti-RSV F-protein antibodies in combination with a diagnosticallyeffective amount of a suitable reporter molecule may be formulated as atest kit for detection of RSV infection.

MATERIALS AND METHODS

[0149] The following Materials and Methods were used in Examples 1 to 6.

[0150] F Protein Preparation and Purification:

[0151] F protein was prepared essentially according to the method ofWalsh et al. J. Gen. Virol, 70, 2953-2961, (1989). Briefly, HEp-2 cellsat 70% confluency were infected with the Long strain of RSV, a labadapted strain of the A type. After culture for 48 hours in T-150culture flasks in IMDM supplemented with 5% fetal calf serum, 2 mMglutamine and 2 mM sodium pyruvate, the cells were lysed in a lysingbuffer of PBS containing 1% Triton X-100 and 1% deoxycholate. F proteinwas purified from the crude cell lysate on an affinity column ofSephadex coupled to a murine monoclonal anti-F antibody, B4 (a kind giftfrom Hiroyki Tsutsumi) (Tsutsumi et al. 1987). The column was washedextensively with lysing buffer and purified F protein was eluted in 0.1°M glycine pH 2.5, containing 0.1% deoxycholate. The eluate wasneutralized immediately with 1 M Tris, pH 8.5 and dialyzed against PBS.After the detergent was removed on a Extracti-D gel column (Pierce,Rockford, Ill., Cat. No. 20346), F protein concentration was determinedby EIA and the solution was sterilized by gamma irradiation.

[0152] Lymphoid Cell Preparation:

[0153] Spleen was obtained following clinically indicated splenectomy ofan idiopathic trombopenic purpura (ITP) patient. A single cellsuspension was prepared by sieving through a metal mesh, and washed inIMDM media supplemented with 2% fetal calf serum. Red blood cells wereeliminated by treatment with ammonium chloride lysing buffer for 90seconds at 37° C. The white blood cell enriched suspension was thenwashed twice with serum containing media, resuspended in ice coldfreezing media (95% FCS with 5% DMSO) at 10⁸ cells/ml and frozen inliquid nitrogen until use.

[0154] In Vitro Immunization (M):

[0155] Cultures were set-up in IMDM supplemented with 2 mM glutamine, 2mM sodium pyruvate, non-essential amino acids, 25 μg/ml IL-2 and 10%fetal calf serum. An antibiotics cocktail was added including 2.5 μl/mlamphotericin, 100 μg/ml ampicillin, 100 μg/ml kanamycin, 5 μg/mlchlortetracycline, 50 μg/ml neomycin and 50 μg/ml gentamicin. The cellswere cultured in 6-well clusters at 3×10⁶ cells/ml with 40 ng/ml Fprotein. After three days, the cells were collected, washed andresuspended in HBSS at 8×10⁸ cells/ml for SCID reconstitution.

[0156] Reconstitution of SCID Mice:

[0157] Five to eight week old female CB17/SCID mice were reconstitutedby intraperitoneal injection of 200 μl of HBSS containing 4×10⁷ humanspleen cells subjected to IVI; the mice were boosted one week later ipwith 5 μg F protein in CFA and tail bled after another 15 days. Theirserum was tested for human IgG concentration and anti-F protein antibodytiter.

[0158] Recovery of Human Cells From hu/SCID Mice:

[0159] Two hu-SPL-SCID mice with high anti-F human antibody titersdeveloped large abdominal tumors. Tumors were recovered by excision fromsacrificed mice under sterile conditions, single cell suspensions wereprepared, the cells were washed with IMDM containing 2% fetal calf serumand cultured at 10⁶/cells ml in T-25 flasks in IMDM with 10% FCS.

[0160] Testing for Human IgG and Anti-F Protein Antibodies:

[0161] The testing for human IgG and anti-F antibodies was performed inELISA. For that purpose, plates were coated overnight with GAH-Ig (0.05μg/well) or F protein (0.05 μg/well) respectively in 0.1 M bicarbonatebuffer, pH 9.5 and blocked with PBS containing 1% fetal calf serum.Serial dilutions of mouse sera, culture supernatants or purifiedantibodies were reacted to the plate. Bound human IgG were revealed bythe subsequent addition of GAH IgG- HRP and OPD substrate (Sigma, . . ., . . . ). Selected high titer human serum was used as a positivecontrol in both assays and purified polyclonal human IgG, or (γ, κ)myeloma protein were used as a standard in the estimation of theconcentration of human IgG and monoclonal antibodies respectively.

[0162] Isotyping of Human Antibodies:

[0163] Isotyping was performed in ELISA on F protein coated plates asdescribed above. Bound human IgG were revealed by the subsequentaddition of HRP conjugated mouse monoclonal antibodies specific forhuman γ1, γ2, γ3, γ4, μ, κ and λ chains. Positive controls were run withmyeloma proteins of the (γ1, κ), (γ2, κ), (γ3, λ), (γ4, λ) or (λ, λ)isotype and free κ and λ chains.

[0164] Protein A Purification:

[0165] Antibodies were purified from culture supernatants on a proteinA-Sepharose 4B column. Briefly, supernatants were collected, filteredthrough 0.2 mm filters and supplemented with 0.02% sodium azide. Columns(gel volume approximately 0.5 ml) were equilibrated in PBS with 0.02%sodium azide, then loaded with supernatant at low speed. After extensivewashing, bound human monoclonal IgG were eluted in 0.1 M sodium citratebuffer, pH 3.5, dialyzed against PBS-azide using Centricon 10 filters(Amicon,) and sterilized by gamma irradiation until further use. Columnswere regenerated with citric acid pH 2.5 and re-equilibrated with PBSwith 0.02% sodium azide for subsequent use.

[0166] Isoelectric Focusing:

[0167] Isoelectric focusing (IEF) of human antibodies was performed inpolyacrylamide pre casted gels (Pharmacia, Uppsala, Sweden, Cat. No.80-1124-80), pH 3-pH 10. Briefly, 20 μl of samples were loaded and runat 1500 volts for 90 minutes. Standards of pi 5.8 to 10.25 were used forpi reference ( . . . ). Gels were stained in Coomassie blue stain anddestained in destaining buffer containing 25% methanol, 68% water and 8%acetic acid.

[0168] Western Blot:

[0169] Purified F protein, both native and denatured by boiling, wasmigrated in a 10% polyacrylamide gel. The gel was blotted on anitrocellulose sheet at 30 volts for 2 hours and 60 volts overnight.After transfer, the nitrocellulose was blocked for 1 hour at roomtemperature with 1% BSA and 0.1% Tween-20 in PBS. Different strips werewashed in PBS and the primary antibodies, hu-SPL-SCID anti-F proteinsera, or hu-SPL-SCID anti-tetanus toxoid negative control, or mouseanti-F protein positive control, were added for 1 hour. All sera werediluted 1:500. After extensive wash with PBS, the secondary antibody,GAH IgG- HRP for the samples and the negative control, or GAM IgG forthe positive control, was added for 1 hour. Blots were revealed with4-chloro-1-naphtol.

[0170] Immunofluorescence:

[0171] RSV infected HEp-2 cells (4×10⁴) were fixed on glass slides usingice cold acetone and were reacted with 20 μl of serum diluted 1:10 orpurified MAb, 2 μg/ml, for 1 hour at 37° C. The slides were washed andthe bound antibodies were revealed with GAH IgG-FITC, for 30 minutes at37° C. and observed under a fluorescence microscope.

[0172] FACScan Analysis:

[0173] RSV-infected HEp-2 cells (10⁶ cells/sample) were washed withwashing buffer (PBS with sodium azide 0.1%). The cell pellet wasresuspended in 50 μl of incubation buffer (PBS with sodium azide 0.1%supplemented with BSA 0.1%) containing 2 μg/ml RF-1 or RF-2. After 15minutes incubation on ice, the cells were washed and resuspended inincubation buffer containing GAH IgG-FITC for another 15 minutes on ice.After 3 washes, the cells were fixed in 1 ml PBS with 1% formaldehydeand analyzed in a Becton-Dickinson FACScan apparatus.

[0174] Affinity Determination:

[0175] Two methods were used to determine the affinity of human MAbs tosoluble F protein:

[0176] In plasmon resonance, using an IASYS machine, antibody was boundcovalently to the wet side of a device from which the change in mass canbe determined based on the change of refraction of light shone on thedry side of the device. Different concentrations of F-protein were addedand subsequently eluted off with a steady flow of PBS. The change inmass as a result of F-protein release from the antibody was measured,and from the kinetics a K_(off) was determined. Ka was calculated bytesting the off-rate from different levels of initial saturation.

[0177] Alternatively, affinity constant was determined bymicro-calorimetry according to Wiseman et al and Robert et al., asfollows: RF-2 and F protein were co-incubated at a known concentrationin a thermo-chamber at 42° C. and the enthalpy change due to the immunecomplex formation in the solution was measured. The reaction wasrepeated at 50° C. The binding association constant K was calculated asa function of temperature and enthalpy change according to Robert et al.in the following equation:

K=Kobs.e ^(ΔHobs/R.(1/T−1/Tobs)) ·e^(ΔCTobs/R.(1/T−1/TTobs))·(T/TObs)^(ΔC/R)

[0178] where Kobs is the binding equilibrium constant and ΔH^(obs) isthe enthalpy change observed experimentally, at a given absolutetemperature, Tobs; R is the universal gas constant (1.987) and ΔC is theexperimentally determined binding heat capacity change.

[0179] Complement-Enhanced Virus Neutralization Assay:

[0180] Two laboratory strains (Long, type A and 18537, type B) and tenwild type RS virus isolates, which were isolated from hospitalizedinfants, were used to assess the neutralizing capacity of anti-F proteinhuman MAbs. Serial dilutions of human MAb were pre incubated with virus(50-100 pfu) in the presence of complement for 30 minutes at roomtemperature, in 100 μl IMDM/well of microtitration plate. HEp-2 cells(5×10⁴/well) were added in 100 μl MEM and incubated for 3 days at 37°C., 5% CO₂. The plates were washed, fixed with acetone and air dried andRSV antigen was detected by ELISA using mouse MAbs. The neutralizationend point was determined arbitrarily as the dilution which reducedantigen production by 50% compared to control wells with no antibody.

[0181] Virus Fusion Inhibition Assay:

[0182] Fusion inhibition titers were determined by pre incubating 100TCID₅₀ RSV Long (prototype A virus) or RS 6556 (Type B clinical isolate)with VERO cells (5×10³/well) in microtitration plates, for 4 hours at37° C., 5% CO₂. Various concentrations of human monoclonal antibodies orcontrols were added to each well and quadruplicate cultures wereincubated for 6 days at 37° C., 5% CO₂. Control cultures contained virusnon infected cells (negative) or infected cells in the absence ofantibody (positive). Virus growth was detected in ELISA using rabbitpolyclonal anti-F protein antisera and HRP-labelled anti-rabbit IgG. Thereaction was developed with TMBlue substrate (KPI, Gaithersburg, Md.).Titers (ED50) were defined as the concentration of antibody inhibitingvirus growth by 50% based on regression analysis of the MAb doseresponse.

EXAMPLE 1

[0183] HU-SPL-SCID Titers:

[0184] Fifteen SCID mice received human spleen cells from a single donorwith ITP condition. The cells were previously cultured for three days inthe presence of IL-2 and different concentrations of soluble F protein.All animals were successfully reconstituted and, after boost with Fprotein, total human IgG concentrations varied from 12 μg/ml to 10 mg/mlin the serum and anti-F protein titers varied from 3×10² to 10⁶ (TableI). No correlation was observed between in vitro F protein exposure andanti-F protein titer in vivo. It has been previously observed with thesubject method, in the horse ferritin antigen system, that antigenexposure is necessary during in vitro cultivation of the spleen cells tosubsequently ensure specific antibody titer in vivo. The discrepancybetween these two systems may be attributed to the difference in theantigens involved: since humans are not naturally exposed to horseferritin, the IVI step involves an antigen priming of the spleen cellsand induces a primary response in vitro; on the other hand, virtuallyall humans are immune to RSV through natural infection in early life,which leads to a permanent memory to F protein, therefore stimulationwith IL-2 alone in vitro followed by one boost in vivo is enough toinduce secondary responses.

[0185] The antisera were polyclonal, as judged from isoelectric focusingpatterns (data not shown). They were tested for reactivity to F proteinin Western blot. Our results showed that polyclonal human Abs didrecognize soluble native F protein both in its dimer form (140 KD) andits monomer form (70 KD); they also reacted strongly with denatured Fprotein, binding specifically to the 2 subunits of 48 KD and 23 KD(representative data in FIG. 1). This suggests that at least a fractionof the humoral response to F protein is directed against linear, nonconformational epitopes of the molecule. Immunofluorescence studiesfurther demonstrated the specificity of the hu-SPL-SCID sera, sinceimmune sera, but not naive SCID mouse sera, reacted strongly withRSV-infected HEp-2 cells (FIG. 2). No reactivity was observed towardsnon-infected HEp-2 cells used as negative control. It was concludedtherefore that soluble F protein was an adequate antigen for thegeneration of antibodies specific to the membrane viral antigenexpressed on naturally infected cells.

EXAMPLE 2

[0186] Identification of Antibodies in Tumor Cell Cultures:

[0187] All mice with high anti-F protein titers were sacrificed andhuman cells were harvested from peritoneal lavage and spleens. Two mice(hu-SPL-SCID # 6 and hu-SPL-SCID #15) spontaneously developed abdominalsolid tumors that were recovered and teased into single cell suspension.The tumor cells secreted specific anti-F protein antibodies asdetermined in ELISA. These tumors and antibodies are referred to as RF-1(RSV F-protein) and RF-2. RF-1 and RF-2 were generated in two differentexperiments separated by approximately two months and were isolated fromindividual hu-SPL-SCID mice, and are thus distinct antibodies; they haveestablished themselves in culture for more than 18 months and 16 monthsrespectively, dividing with an approximate doubling time of 36-48 hours.Specific antibody concentration is typically of 0.5-1 μg/ml in a cultureseeded at 0.5×10⁶ cells/ml and grown for three days.

[0188] For further characterization, both human MAbs were purified fromculture supernatants by affinity chromatography, using Protein ASepharose columns. Both RF-1 and RF-2 are IgG(_(l,k)), with half maximalbinding to F-protein in ELISA at 0.6 and 1 mg/ml respectively (FIG. 3).From the migration pattern in IEF, RF-1 and RF-2 isoelectric points weredetermined to be 8.8 and 8.9 respectively (FIG. 4). RF-1 and RF-2specifically recognized RSV infected HEp-2 cells in flow cytometry (FIG.5). The dissociation constant, Kd, for RF-1 was determined by plasmonresonance on an IASYS machine to be in the 10⁻¹⁰ M range. The Kdconstant of RF-2 was determined by titration micro calorimetry,according to Wiseman et al (1989) and Robert et al. (1989) to be 2×10⁻⁹M.

EXAMPLE 3

[0189] Tissue Specificity of Anti-F-Protein:

[0190] Purified antibodies were screened for reactivity to a series ofhuman cell lines available at ATCC by means of indirectimmunofluorescence assays measured by flow cytometry (Table II): Theresults showed that the antibodies did not bind to cell linesrepresenting respiratory tract lining (HEp-2, a laryngeal epidermoidcarcinoma, Cat. No. CCL 23), liver (HepG2, a human hepatoma cell line,Cat. No. HB 8065), lymphoid tissue (SB, a human B lymphoblastoid cellline, Cat. No. CCL 120 and HSB, a T lymphoblastoid line, cat. no. CCL120.1) and prostate (LNCaP.FGC, a human prostate adenocarcinoma line,Cat. No. CRL 1740).

EXAMPLE 4

[0191] In Vitro Functional Activity:

[0192] To determine whether the antibodies had virus neutralizing effectin vitro, they were subjected to two types of functional assays:Infection neutralization assays were performed by pre-reacting the viruswith purified MAb prior to its addition to the cells and thereforereflect the ability of the MAb to inhibit virus infectivity; fusioninhibition reflects the ability of the Ab to inhibit virus growth andexpansion after virus entry in the cell. The outcome of both assays wasmeasured as the amount of virus released in the culture after a givenincubation time, as determined by viral antigen titration in EIA.

[0193] Both Abs were able to inhibit virus infection, of all twelveisolates tested, at concentrations ranging from 30 ng/ml to 1000 ng/mland from 8 ng/ml to 165 ng/ml, for RF-1 and RF-2 respectively. RF-2performed consistently better than RF-1, yielding to 50% virusinhibition (ED50) at concentrations 1.25 to 10 times lower than RF-1.Representative data are indicated in Table III.

[0194] As expected, higher concentrations of MAb were required toinhibit fusion and viral antigen expression in previously infectedcells. In this assay, RF-1 was 5 to 10 times more potent than RF-2. BothMAb were more effective in the Type B prototype RS 6556 than in the TypeA prototype RS Long (Table III). TABLE I hu IgG mouse # [Ag] in vitrofresh cells (μg/ml) anti-F titer 1 1 μg/ml + 1,000 10⁶ 2 1 μg/ml + 12.310³ 3 1 μg/ml + 3,000 10⁶ 4 1 μg/ml + 8,750 10⁶ 5 1 μg/ml + 1,000 10⁶ 61 μg/ml − 1,500 10⁵ 7 1 μg/ml − 162 10⁵ 8 1 μg/ml − 4,500 10⁶ 9 1 μg/ml− 333 10⁵ 10 40 ng/ml − 3,300 5 × 10⁵ 11 40 ng/ml − 554 3 × 10² 12 1μg/ml − 10,000 5 × 10⁵ 13 1 μg/ml − 200 5 × 10⁴ 14 0 μg/ml − 182 5 × 10⁴15 0 μg/ml − 3,300 10⁵

[0195] Table I: Splenocytes from a single donor were cultured in thepresence of IL-2 for 3 days, with or without F protein. SCID mice werereconstituted with 4×10⁷ cells and boosted with 10 μg of F protein ip inCFA. In mice # 1, 2, 3, 4 and 5, fresh autologous cells (20×10⁶) wereinjected with the boost. Human IgG concentration was determined bycomparison to a standard curve of polyclonal IgG and anti-F proteintiter was determined by end point dilution in EIA. TABLE II Cell lineTissue Type Tissue Labeling HEp-2 Laryngeal epidermis − RSV infected-HEp-2 Laryngeal epidermis (RSV) ++++ SB Lymphoid − HSB Lymphoid − LNCaPProstate − HepG2 Liver −

[0196] Table II: Reactivity of RF-2 with various cell lines. Variouscell lines were subjected to indirect immunofluorescence labeling withRF-2, 200 ng/10⁶ cells. A Fab goat anti-human IgG-FITC was used assecond step. (−) indicates the presence of RF-2 did not result in changeof channel for the average fluorescence; (+) indicated increase ofaverage labeling by 0.5 log. TABLE III Fusion Inhibition activityInfection Neutralization ED₅₀ titer Activity ED₅₀ titer Anti- RS Long RS6556 Anti- MR 144 body (Type A) (Type B) body (Type A) 18537 (Type B)RF-1  660 ng/ml  40 ng/ml RF-1 30 ng/ml 30 ng/ml RF-2 3300 ng/ml 400ng/ml RF-2  8 ng/ml 12 ng/ml

[0197] Table III: ED₅₀ is defined as concentration of antibodyinhibiting virus growth by 50% based on regression analysis of themonoclonal antibody dose-response.

EXAMPLE 5 (COMPARATIVE)

[0198] Induction of IgG Recall Responses to F-Protein In Vitro:

[0199] More than 95% of the population over 2 years of age have beenexposed to, and responded successfully to RSV Henderson et al. J. Med.(1979), 300, 530-534. Challenge of spleen cell in vitro with RSVF-protein should, therefore, result in recall responses, and, indeed,mainly IgG responses were induced in vitro with spleen cells (see FIG.5). The optimal antigen concentration, 40 ng/ml, was at least one orderof magnitude lower than what was observed for antigens inducing primaryresponses, i.e. ferritin, Ilig/ml, Boerner et al, J. Immunol., 1991,147, 86-95; Brams et al, Hum. Antibod. Hybridomas, 1993, 4, 47-56.Therefore, it must be considered that in vitro priming with F-proteininduces secondary like responses. Several attempts to induce significantin vitro responses to RSV F-protein failed with PBMCs and tonsil derivedcells.

[0200] A limited effort to generate monoclonal antibodies from in vitroprimed spleen cells resulted in several monoclonal IgG antibodies to RSVF-protein. Most of these, however, cross-reacted to one of severalcontrol antigens in ELISA (results not shown).

EXAMPLE 6

[0201] Cloning of the Genes Coding for RF-2:

[0202] Neither the RF-1 nor the RF-2 clone produce significant amountsof antibody. Also, both of these cell lines grow best in media with 20%FCS, which is disadvantageous because it results in contamination of thepurified antibody with bovine IgG. Therefore, in order to be able toproduce and purify amounts of antibody necessary for doing meaningfulanimal model tests, which typically requires up to 1 gram of oneselected antibody, it is advantageous to transfer the genes coding forRF-1 and RF-2 to a production vector and cell line. The presentassignee, IDEC Pharmaceuticals, Inc., has developed a very efficienteukaryotic production system which results in the production of humanmonoclonal antibodies in CHO cells. This vector system is described incommonly assigned U.S. Ser. No. 08/379,072, filed Jan. 25, 1995, and incommonly assigned U.S. Ser. No. 08/149,099, filed Nov. 3, 1993, both ofwhich are incorporated by reference herein. Routinely using this systemantibody gene transfected CHO cells produce around 200 mg antibody perliter of serum free medium in spinner cultures and greater than 500mg/liter in fermentors after amplification in methotrexate.

[0203] Cell culture cloned (see below) RF-2 cells, approximately 5×10⁶,were subjected to RNA extraction using a mRNA isolation kit, Fast Tract(InVitroGen, San Diego, Calif.), and single stranded cDNA was preparedusing an oligo-dT primer and reverse transcriptase. An aliquot of cDNAwas used as the starting material for polymerase chain reaction (PCR)amplification of the variable region genes. PCR was performed using twosets of primers. (see Table IV). TABLE IV* Heavy chain primers with Mlu1 Site V_(H)1 5′ (AG)₁₀ ACGCGTG(T/C)CCA(G/C)TCCCAGGT(G/C)CAGCTGGTG 3′V_(H)2 5 (AG)₁₀ ACGCGTGTC(T/C)TGTCCCAGGT(A/G)CAG(C/T)TG(C/A)AG 3′ V_(H)35 (AG)₁₀ ACGCGTGTCCAGTGTGAGGTGCAGCTG 3′ V_(H)4 5 (AG)₁₀ACGCGTGTCCTGTCCCAGGTGCAG 3′ V_(H)5 5 (AG)₁₀ACGCGTGTCTGGCCGAAGTGCAGCTGGTG 3′ Heavy chain constant region primeranti-sense strand with Nhe 1 site IgG1-4(AG)₁₀GCCCTTGGTGCTAGCTGAGGAGACGG 3′ Kappa Chain primers with Dra IIIsite 1. 5′ (AG)₀₁CCAGGTGCACGATGTGACATCCAGATGACC 3′ 2. 5′(AG)₀₁CCTGGATCACGATGTGATATTGTGATGAC 3′ 3. 5′(AG)₀₁CCAGATACACGATGTGAAATTGTGTTGAC 3′ 4. 5′(AG)₁₀TCTGGTGCACGATGTGACATCGTGATGAC 3′ Kappa constant region primeranti-sense strand with Bsi WI site C_(k) 5(AG)₁₀TGCAGCCACCGTACGTTTGATTTCCA(G/C)CTT 3′

[0204] The first set of primers was designed for amplifying the heavychain variable regions. It consists of one 3 primer that binds in the Jregion and five family-specific 5′ primers that bind in the late leaderand framework 1 region. A second set of primers was designed foramplifying the Kapp variable region. It consists of one 3′ primer andfour 5′ primers that bind in the late leader and framework 1 regions.The PCR reactions were electrophoresed on agarose gels and correctlysized 350 base pair bands were excised. The DNA was electroeluted, cutwith appropriate restriction enzymes and cloned into IDEC's NEOSPLAexpression vector. (See FIG. 6) The NEOSPLA vector used for expressionof human antibodies contains the following: CMV=cytomegaloviruspromoter, BETA mouse beta globin major promoter, BGH=bovine growthhormone polyadenylation signal, SVO=SV40 origin of replication.N1=Neomycin phosphoamsferase exon 1, N2=Neomycin phosphotransferase exon2. LIGHT=Human immunoglobulin kappa constant region. Heavy=Humanimmunoglobulin gamma 1 or gamma 4 PE constant region. L=leader. SV=SV40polyadenylation region.

[0205] IDEC's NEOSPLA expression vectors were designed for large scaleproduction of immunoglobulin genes (See, Reff et al, Blood, (1994), 83,435-445, incorporated by reference in its entirety). Mouse/humanchimerics, primate/human chimerics and human antibodies have beensuccessfully expressed at high levels using these vectors. NEOSPLAcontains a neomycin phosphotransferase gene for selection of CHO cellsthat have stably integrated the plasmid vector DNA. In addition, NEOSPLAcontains a dihydrofolate reductase gene for amplification inmethotrexate, a human constant light chain (either K or X) and a humanconstant heavy chain region (either γ1 or γ4(PE)). Gamma 4 (PE) is thehuman γ4 constant region with 2 mutations, a glutamic acid in the CH2region which was introduced to eliminate residual FcR binding, and aproline substitution in the hinge region, intended to enhance thestability of the heavy chain disulfide bond interaction, Algre et al, J.Immunol., 148, 3461-3468, (1992); Angal et al, Mol. Immunol., 30,105-108 (1993), both of which are incorporated by reference herein.Unique restriction sites have been incorporated into the vector in orderto facilitate insertion of the desired and light variable regions. Reffet al., Blood, (1994), 83, 435-445.

[0206] The light chain of RF-2 has been cloned into NEOSPLA in duplicateand sequenced following the method of Sanger et al. Sanger et al., Proc.Natl. Acad. Sci. (1977), 74, 5463-5467. The kappa chain is a member ofthe kappa 2 subgroup. Similarly, the human heavy chain variable regionof RF-2 has been isolated and cloned in front of the human γ1 constantdomain.

[0207] The light chain coding genes of RF-1 and RF-2 were readilycloned, whereas cDNA for the genes encoding the heavy chains could notbe generated using the common Tac reverse transcriptase. However, thisproblem was obviated by substituting a high temperature, 70° C., reversetranscriptase. Thereby, intact PCR products could be generated withprimers primarily derived from V_(H)2 family genes.

[0208] The amino acid sequence and the nucleic acid sequence for theRF-1 light and heavy variable domains may be found in FIGS. 7a and 7 b,respectively. The amino acid sequence and the nucleic acid sequence forthe light and heavy variable domains for RF-2 may be found in FIGS. 8aand 8B, respectively. FIGS. 9a-9 c depict the nucleic acid and aminoacid sequence of RF-1 as expressed in the subject NEOSPLA vector. FIGS.9a and 9 b depict the leader, variable light and heavy, and humanconstant domain sequences, i.e., the human kappa domain and the humangamma/constant domain. FIG. 9c shows the amino acid and nucleic acidsequence of the human gamma/constant domain. FIG. 10 depictsschematically an expression vector which provides for the expression ofthe sequences set forth in FIGS. 9a-9 c and thereby recombinant RF-1 inCHO cells.

[0209]FIGS. 11a-11 a similarly depict the amino acid and nucleic acidsequences of the leader sequence, RF-1 variable light, human kappaconstant region, RF-2 variable heavy, and human gamma/constant domain.FIG. 12 depicts schematically an expression vector which provides forthe expression of recombinant RF-2 in CHO cells.

EXAMPLE 7

[0210] Development of a Protocol for Cloning of EBV Transformed Cells:

[0211] Antibody production from EBV transformed cells continuouslydecrease, and ultimately ceases. Kozbor et al., J. Immunol. (1981), 127,1275-1280. To immortalize the antibody production, it is thereforeessential to extract the immunoglobulin coding genes from the cellsbefore this event and transfer those into an appropriate expressionsystem. In order to isolate the genes coding for the antigen bindingvariable domains of antibodies produced by EBV transformed cells, it isessential to ensure that the cell material is monoclonal. EBVtransformed cells are, however, very difficult to clone whether bylimiting dilution or in semisolid agar. Isoelectric focusing gelelectrophoresis of protein A purified preparations of our two anti-Fprotein antibodies, RF-1 and RF-2, showed at least two populations ofantibodies in the RF-2 preparation and the possibility of oligoclonalityin the RF-1 preparation.

[0212] By using the mouse thyoma line EL-4 B5 Zhang et al, J. Immunol.,(1990), 144, 2955-2960, as feeder layer, cells were expanded from asingle cell through limiting dilution. The human thyoma cell line EL-4B5 expresses gp39 in a membrane receptor way that induces B cells togrow. 5×10⁴ EL-4 B5 cells/well were plated out in a microliter plate,and cells from the cultures were plated out on the EL-4 B5 layer atvarious concentrations, from 0.38 cells/well and up. The number of wellswith growth for each of the concentration plated were counted after anappropriate amount of time.

[0213] The supernatant was tested for presence of human IgG and forantigen-specific IgG. With this protocol we have isolated and cloned thecells that produce RF-1 (see Table V) and RF-2 (see Table VI),respectively, from the original oligoclonal preparations. Thenon-specific antibodies found in the cloning were only analyzed withrespect to isotype, and were found to be the same as the specificantibodies, IgGlk. Based on the yield of F-protein specific clones fromfreezes made at various time points during the cultivation of RF-1, aswell as the amount of IgG that was produced, it was estimated that thespecific antibody made up approximately {fraction (1/20)} of the totalantibody amount shortly after the start of the culture and disappearedafter approximately 8 months in culture. RF-2 made up a much higher partof the total IgG, no less than 10% at any given time. Antibody from theoligoclonal preparations was used to generate the in vitroneutralization data, resulting in an overestimation of the ED50 titers.Our affinity studies with plasmon resonance, however, were not dependenton using pure antibodies. The affinity studies using titration microcalorimetry was done with cloned material. TABLE V* # cells/well # wells# anti-F wells (%) # wells with growth (%) 30 48 48(100) 48(100) 10 4848(100) 48(100) 3.3 96 27(28)  68(71)  1.1 192 17(9)  112(58)  0.38 38418(5)  116(30) 

[0214] TABLE VI* # cells/well # wells # anti-F wells (%) # wells withgrowth (%) 30 40  40(100) 15(37.5) 10 120 120(100) 22(18) 3.3 120102(85)  9(7.5) 1.1 120  50(41.6)  1(0.83) .33 180  30(16.7)  5(2.8)

[0215] In order to confirm the clinical applicability of the two humanmonoclonal antibodies with in vitro virus neutralizing activity, theseantibodies are further characterized with respect to their efficacy inclearing RSV infection in two different animal models. These preclinicalperformance evaluations are effected with material produced by CHO cellstransfected with the cloned genes coding for the antibodies insertedinto a proprietary expression vector (see FIG. 6). Two antibody models,one with intact complement and Fc receptor binding domains, γ1, and onevoid of these domains, γ4 (PE mutant), Alegre et al., J. Immunol.,(1992), 148, 3461-3468; Angal et al., Molecular Immunology, (1993), 30,105-108, will be tested. The rationale for testing γ4 version is basedon two considerations: (i) Anti-F-protein Fabs have shown significantvirus neutralizing effect in vitro Barbas et al., Proc. Natl. Acad. Sci.(1992), 89, 10164-10168, as well as in vivo, Crowe et al, Proc. Natl.Acad. Sci. (1994), 91, 1386-1390, albeit when administered directly intothe lung, (ii) potentially avoiding lung damage caused by effectorfunction activation in sensitive tissue already stressed by virusinfection could be advantageous. A set of nonspecific controlantibodies, one γ1 and one γ4 (PE), will be generated from an in-housenonspecific hybridoma IgG₁ antibody.

[0216] The first animal model is a mouse model, Taylor et al., J.Immunology (1984), 52, 137-142; Walsh, E. E., J. Infectious Diseases(1994), 170, 345-350. This model is used to determine the effectivedose, defined as the smallest dose resulting in a 2 log reduction invirus load in the lung tissue after 1 weeks incubation. This model isalso used to determine which of the antibody models to proceed with. Thesecond animal model is a primate model using the African green monkey,Kakuk et al, J. Infectious Diseases (1993), 167, 553-561. RSV causeslung damage in the African green monkey, and this model's main purposeis for evaluating the damage preventing properties of the antibodies.The number of tests with this model will be limited to test one antibodyin 5 different doses. The antibody, the dose and the infusion daterelative to infection date will be determined based on the findings withthe mouse model. Lung section is examined for virus load in plaque assayand by light microscopy for detection of lesions caused by RSV.

[0217] It will be observed if any changes in the amino acid sequencehave taken place during the process of stimulating and expanding thecells that produce the two antibodies. This is done by testing with aset of PCR primers based on the CDR3 regions of the heavy chains of RF-1and RF-2 whether the sequences of the genes coding for RF-I and RF-2 arepresent in the original frozen cell material from which the two celllines were generated. The positive control is RF-1 and RF-2 spikedsource cells. The analysis follows the principles established by Levy etal., 1989, Levy et al., J. Exp. Med. (1989), 169:2007 and Alegre et al.,J. Immunol. (1992), 148, 3461-3468; Angal et al., Molecular Immunology(1993), 30, 105-108; Foote et al., Nature (1991), 352, 530-532; Rada etal., Proc. Natl. Acad. Sci. (1991), 88, 5508-5512; Kocks et al., Rev.Immunol. (1989), 7, 537-559; Wysocki et al., Proc. Natl. Acad. Sci.(1986), 83, 1847-1851; Kipps et al., J. Exp. Med. (1990), 171, 189-196;Ueki et al., Exp. Med. (1990), 171, 19-34.

EXAMPLE 8

[0218] Generate CHO Cell Lines That Produce Large Amounts (>100mg/Liter) of RF-1 and RF-2:

[0219] a. Transfect Expression Plasmids, Isolate and Expand G418Resistant CHO Clones Expressing the Highest Levels of RF-1 and RF-2.

[0220] Once the RF-1 and RIF-2 variable region genes are cloned intoNEOSPLA, Chinese hamster ovary (CHO) cells (DG44), Urlaub et al, J.Somat. Cell Mol. Genet., (1986), 16, 555, are transformed with theplasmid DNA. CHO cells are grown in SSFM H minus hypoxanthine andthymidine (G1BCO). 4×10⁶ cells are electroporated with 25 μg plasmid DNAusing a BTX 600 electroporation device (BTX, San Diego, Calif.) in 0.4ml disposable cuvettes. Prior to electroporation, the plasmid DNA willbe restricted with Pac I which separates the genes expressed inmammalian cells from the portion of the plasmid used for growth inbacteria. Conditions for electroporation are 230 volts, 400 microfaradays, 13 ohms. Each electroporation is plated into a 96 well dish(about 40,000 cells/well). Dishes are fed with media containing G418(Geneticin, GIBCO) at 400 μg/ml three days following electroporation,and thereafter, periodically, until colonies arise. Supernatant fromcolonies is assayed in ELISA for the presence of human IgG and foranti-F-protein activity.

[0221] b. Amplify the Expression of Antibody in Methotrexate.

[0222] The G418 resistant colonies producing the highest amount ofimmunoglobulin are then transferred to larger vessels and expanded. Theexpression of the highest secreting G418 clone is increased by geneamplification, by selection in 5 nM methotrexate (MIX) in 96 welldishes. The 5 nM colonies producing the highest amount of antibody arethen expanded and then expression amplified again by selection in 50 nMMTX in 96 well dishes. Following this protocol, we have previously beenable to derive CHO cells that secrete greater then 200 mgs/liter in 7days in spinner culture (greater than 0.5 gram/liter in fermentors in 6days). Human antibody is then purified from supernatant using protein Aaffinity chromatography.

[0223] c. Produce and Purify Antibody.

[0224] 100 mg of each antibody is generated. The selected antibody isproduced in amounts determined from the mouse model studies. Spinnerflasks with selected CHO transfectomas in CHO—S SFM II serum free medium(GIBCO Cat. No. 91-0456DK) with 50 nM methotrexate are used to produceantibody in the required amounts. Supernatant are harvested and filteredthrough a set of filters to remove particular material, ending up with a0.2 nm filter. The supernatant is run through a protein A column with apredetermined size based on the total amount of antibody. After washing,the antibody is eluted from the column with 0.1 M Glycine/HCl, pH 2.8,into a neutralization buffer, 1 M Tris./HCl, pH 7.4. Theelution/neutralization buffer is exchanged extensively, ≧1000 times,with sterile PBS by ultrafiltration through an Amicon Centriprep orCentricon 30 (Cat. no. 4306 and 4209). The concentration of antibody isadjusted to 2 mg/ml and sterilized by filtration through a 0.2 nmfilter. The antibody is purified and stored on ice in 2 ml cryotubesuntil use in animals.

EXAMPLE 9

[0225] Characterize RF-1 and RF-2 in Respect to Performance in RSVAnimal Models:

[0226] The performance of RF-1 and RF-2 is determined using appropriateanimal models. The evaluation is divided into two steps, first (a) aBalb/c model, Taylor et al., J. Immunology (1984), 52, 137-142; Crowe etal., Proc. Natl. Acad. Sci. (1994), 91, 1396-1390; Connors et al., J.Virology (1992), 66, 7444-7451, to determine the potency of theantibodies, as well as to determine what type of support (effector)functions are essential for the antibody to clear the virus load. Fromthe data gained in the mouse model, one candidate is chosen for furtherstudies in a primate model. The primate model is an African green monkeymodel, Kakuk et al., J. Infectious Diseases (1993), 167, 553-561. Aprimate model is especially suitable for confirming that the subjectmonoclonal antibodies can be used to prevent virus associated lungdamage.

[0227] a. Test Performance in Mouse Model.

[0228] The rodent-model we have chosen is the Balb/c mouse. This modelis well characterized, Crowe et al., Proc. Natl. Acad. Sci. (1994), 91,1386-1390; Connors et al., J. Virology (1992), 66, 7444-7451, forstudies on passive therapy studies. Balb/c mice are highly permissive togrowth of RSV in both upper and lower airways at all ages, Taylor etal., J. Immunology (1984), 52, 137-142. Animals are housed in groups of5, fed standard mouse chow and water ad libitum and cared for accordingto the Rochester General Hospital vivarium guidelines. These guidelinesare in compliance with the New York State Health Department, the FederalAnimal Welfare Act and DHHS regulations. All procedures, includinginjections, virus infection, orbital bleeding and sacrifice by cervicaldislocation, are performed under penthrane anesthesia in a vented hood.

[0229] i. Determine Effective Dose of Antibody and Compare Performanceof γ1 and γ4 (PE Version) of RF-I and RF-2.

[0230] Groups of 5 mice are infected by intranasal instillation of 106Long (subgroup A) or 10⁵ 18537 (subgroup B) plaque forming units (PFU)of RSV in a 100 μL volume on day 0. On day four, at peak virus titer,animals will be injected intraperitoneally with each of the fourF-protein specific monoclonal antibody preparations or control antibody.The doses tested are initially centered around a reference dosecalculated to provide a serum neutralization titer of approximately1:300 or greater from in vitro studies. This titer has been associatedwith protective levels against challenge with RSV in small animals. Doseresponse is evaluated by treatment with 25, 5, 1, ⅕ and {fraction(1/25)} of the reference dose. Experiments with higher or lower dosesare performed if warranted. Control mice are injected with an equivalentdose of the isotype matched monoclonal antibodies, as described above.Twenty-four hours later; day 5 is the peak of virus shedding, the miceare sacrificed. Serum is obtained by intracardiac puncture and the nasalturbinates and lungs are removed, weighed and homogenized in 1 and 2 mlof NMM, respectively. Homogenates are titered for virus on HEp-2 cells,and virus titers expressed as TCID₅₀/gm tissue. The mean titers betweengroups are compared to the control group by the student t-test. Serum isobtained at the time of infection and at sacrifice for human monoclonalantibody quantification by enzyme immunoassay and neutralization assay.It is anticipated that the greatest reductions in virus titer will be inlung virus growth since IgG isotypes are not actively secreted (incontrast to IgA) in the upper respiratory tree. Should therapy on day 4of infection prove ineffective at reducing lung virus, therapy on days 2and/or 3 will be assessed.

[0231] The titer of each monoclonal antibody in stock solutions and inserum from injected animals is determined using an ELISA, as describedsupra. RSV fusion protein purified by affinity chromatography accordingto established methods (82) is used in the solid phase. A separate assayfor the RSV G protein will also be devised for evaluation of mouse IgGresponses to experimental RSV infection. Rabbit antibody specific forhuman IgG and mouse IgG (available from Virion Systems, Inc., Bethesda,Md.) is used to detect human monoclonal antibody or mouse antibody inthe ELISA.

[0232] The dose response effect of the monoclonal antibodies aredetermined for the antibodies as the lowest antibody titer which reducesvirus titers more than 2 log₁₀ or >99% reduction in virus titer. Thedegree of protection are correlated to the serum antibody levelsachieved at the time of sacrifice. In addition, the potentialsynergistic effect of various combinations of human monoclonalantibodies is determined. The results of the initial in vitro studiesoutlined above will be used to guide the in vivo experiments. Forinstance, if RF-1 and RF-2 have distinct antigenic binding sites on theRSV F protein, combinations of the same isotype (γ1 or γ4) may providefor synergistic protection.

[0233] ii. Histological Evaluation of Lung Tissue.

[0234] The effect of passive therapy on lung inflammation is evaluatedby standard histopathological and immunohistochemical techniques. Bothperibronchiolar infiltrates and alveolar infiltrates have been describedin the mouse following either primary or secondary infections, Connerset al, J. Virology, (1992), 66, 7444-7451. Experimental animals aretreated with monoclonal antibody, as described above. Uninfecteduntreated control mice serve as comparisons for evaluating histologicaleffects. On days 5 and 8 after infection, the lungs are removed andinflated with formalin under constant filling pressure (30 cm H₂O) for30 minutes. After sectioning and staining with hematoxylin-eosin, thedegree of inflammatory infiltrate (PMN and lymphocytic separately) inthe peribronchiolar and alveolar areas is determined using astandardized scoring system. Since it is anticipated that the γ1 and γ4monoclonal antibodies may fix and activate complement differentially,the lung sections are stained for mouse C3 deposition in areas ofinflammation using a commercially available rabbit anti-mouse C3antibody (Viron Systems, Inc. Bethesda, Md.) and peroxidase conjugatedgoat and-rabbit IgG.

[0235] In addition to evaluation of histological changes seen in fixedpulmonary tissues, pulmonary inflammation is assessed by evaluation ofalveolar cytology. Groups of mice, treated as described above, aresacrificed and a bronchoalveolar lavage (BAL) performed by repeatedlyinfusing 3 ml PBS into the lower airway. Cell counts of the BAL will beperformed, and the cell type identified by staining of cytocentrifugepreparations.

[0236] iii. Effect of Antibody Therapy on the Natural Immune Response toInfection.

[0237] In the cotton rat and owl monkey models, passive therapy of RSVinfection with polyclonal IgG preparations diminishes the subsequentnatural antibody response to the virus, although animals are fullyprotected upon re challenge, Hemming et al., J. Infectious Diseases(1985), 152, 1083-1086; Prince et al., Virus Research (1985), 3,193-206. In contrast, Graham found that treated mice had both bluntedantibody responses and were susceptible to virus re challenge, Graham etal., Ped. Research (1993), 34, 167-172. To assess this possibility usinghuman monoclonal antibodies, mice are infected with the Long strain ofRSV and treated with a protective dose of antibody on day 4, as outlinedabove. Controls will include infected untreated animals, and uninfectedtreated animals. Mice are bled for antibody determination every otherweek for 8 weeks and then every 4 weeks for an additional 8 weeks. Bothhuman monoclonal antibody and mouse antibody to the RSV F and G proteinsare determined by ELISA. In addition, the neutralization titer of theserum determined at each time point. The contributions to neutralizationby the residual human monoclonal antibody and actively produced mouseantibody are inferred from the ELISA results and by the results ofneutralizing activity of the uninfected antibody treated controls. Whenhuman monoclonal antibody is undetectable by ELISA, animals arerechallenged with the same strain of RSV. After 4 days, the animals aresacrificed and the lungs and nasal tissues titered for virus andcompared to control groups.

[0238] In order to assess the impact of monoclonal antibody therapy oncytotoxic T-cells (CTL) induction, similar experiments are carried, butsix weeks after infection, mice are sacrificed and spleen cell culturesstimulated with live RSV for 5 days, Walsh, E. E., J. InfectiousDiseases (1994), 170, 345-350. CTL activity is assessed by standardChromium 51 release assay using persistently infected Balb/c fibroblastcell line (BCH4 cells) and compared to an uninfected Balb/c fibroblastline.

[0239] Based primarily on the effective dose studies of passive therapyof established RSV infections, it is then determined which antibody,RF-1 or RF-2, is the most efficacious for preventing or treating RSVinfection. The choice between the γ1 or γ4 (PE) versions takes the lunghistology studies into account, in particular whether recruitment ofcomplement appear to be significantly enhanced with the Clq bindingantibody. Massive activation of complement could potentially haveadverse effects, although enhanced vascularization that follows mightincrease the virus-antibody confrontation.

[0240] c. Test Performance in Monkey Model.

[0241] The decisive test for the selected antibody is in a primatemodel. We have chosen the African green monkey (Cercopithecus aethiops)because it is highly permissive for RSV, and infection leads to enhancedlung pathology and detectable lesions, Kakuk et al., J. InfectiousDiseases (1993), 167, 553-561. African green monkeys are also readilyavailable and not endangered. This monkey weighs between 5 and 10 kgs.The highest expected maximal dose of antibody is 20 mgs/kg. Threeanimals of each 10 kgs with 20 mgs/kg equals 600 mgs of antibody. Somewild African green monkeys are naturally immune to RSV, and arequirement for entering monkeys into our study is that they are serumnegative to RSV.

[0242] Based on the baseline established in the mouse model, effectivedose/kg and infection time prior to therapy, a limited series of testsare performed in order to establish effective dose for virus reduction,as well as to confirm whether this correlates with prevention of lungpathology, in particular parenchymal inflammatory involvement. Only onevirus strain, Long (subtype A) is tested. Initially 25, 5, 1, and{fraction (1/25)} times the reference dose is tested. Two controlgroups, one that receive virus but no antibody, and another thatreceives virus and maximal dose of the isotype matched control antibody,are analyzed. Essentially, the experiments are effected as describedabove. Monkeys in groups of 3 are also infected by intranasalinstillation with 10⁶ PFU of virus. Six to seven days after infectionwith virus the monkeys are sacrificed and lung and pharynx samples aretaken for viral assays as described above and for histology.

[0243] Histology are performed essentially, as described above. Briefly,the lungs are perfused with 10% neutral buffered formalin under constantfilling pressure. The lungs will remain in formalin for at least oneweek. After sectioning and staining with hematoxylin-eosin, the slidesare evaluated histopathologically according to Kakuk et al., J.Infectious Diseases (1993), 167, 553-561. Serum samples are also betaken in order to determine the titer of human are antibody to RSV inELISA and in Infection Neutralization assays.

EXAMPLE 10

[0244] 1. Confirm Tissue Specificity by In Vitro Test on Human TissueSections.

[0245] The antibody is, then further tested for potential crossreactivity to normal tissues by immunohistology studies on differentfrozen normal tissue sections from two different individuals. Briefly,Cryostat microtome cuts of frozen tissues are subjected to 3 tests:Fixation analysis, a Nitration analysis and a specificity/distributionanalysis Purified biotin labeled anti-RSV F-protein antibody in PBS with1% BSA is added, and the slide is incubated for 30 min. in a humidifiedchamber at 200C. The slide is then washed in PBS with 1% BSA. The slideis subsequently incubated with Avidin-HRP in PBS with 1% BSA for 30 min.HRP is allowed to react with 3,3 diaminobenzidine-tetrahydrochloride,which forms an insoluble precipitate stain mediated by oxidation withHRP. This will identify any potential cross reactions of the subjecthuman monoclonal antibodies. This test will be performed by ImpathLaboratories, N.Y., N.Y, and is approved by the FDA for I.N.D.submissions for products destined for human therapy. This histologyapproach uses pre-existing tissue and is less costly than thealternative, targeting studies of RSV infected monkeys with radiolabeledantibody.

1. A human monoclonal antibody which specifically binds the RSV fusionprotein and which possesses an affinity for the RSV F-protein of ≦2×10⁻⁹molar.
 2. The human monoclonal antibody of claim 1 which neutralizes RSVin vitro.
 3. A human monoclonal antibody which specifically binds to theRSV fusion protein which is selected from the group consisting of RF-1,RF-2 and recombinant human monoclonal antibodies which contain thevariable heavy and light domains of either RF-1 or RF-2.
 4. The humanmonoclonal antibody of claim 3 wherein said antibody is RF-1.
 5. Thehuman monoclonal antibody of claim 3 wherein said antibody is RF-2. 6.The human antibody of claim 3, wherein said antibody is a recombinantantibody which contains either the human gamma 1, human gamma 4, orhuman gamma 4 PE constant region.
 7. Eukaryotic cells which have beentransfected with DNA sequences which encode for the heavy and lightvariable domains of either RF-1 or RF-2.
 8. The cells of claim 7 whereinsaid eukaryotic cells are CHO cells.
 9. The eukaryotic cells of claim 7wherein said DNA sequences are selected from the DNA sequences set forthin any one of FIGS. 7a, 7 b, 8 a, 8 b, 9 a, 9 b, 10 a or 10 b.
 10. AnEpstein-Barr immortalized B cell line which secretes a human monoclonalantibody which possesses an affinity for the RSV fusion protein of≦2×10⁻⁹ molar.
 11. The cell line of claim 10 wherein said antibodyneutralizes RSV in vitro.
 12. The cell line of claim 10 wherein saidcell line is selected from the group consisting of RF-1 and RF-2.
 13. Amethod for producing human antibodies specific to the RSV fusion (F)protein which comprises: (i) priming human splenocytes in vitro in thepresence of IL-2; (ii) transferring said primed human splenocytes into aSCID mouse; (iii) boosting said SCID mouse with RSV F-protein; and (iv)isolating human B cells from said SCID mouse which secrete humanmonoclonal antibodies specific for the RSV F-protein.
 14. The method ofclaim 13 wherein said isolated human B cells are immortalized.
 15. Themethod of claim 14 wherein immortalization is effected usingEpstein-Barr Virus (EBV).
 16. The method of claim 15 wherein said EBVimmortalized cells are cloned using the mouse thyoma cell line EL-4 B5as a feeder layer.
 17. The method of claim 13 wherein the priming stepis effected in the presence of IL-4 or IL-6.
 18. A DNA sequence whichencodes for the variable heavy and/or variable light domain of RF-1 orRF-2.
 19. An expression vector which provides for the expression of aDNA sequence according to claim
 18. 20. The DNA sequence of claim 18which is selected from the group consisting of the DNA sequences setforth in FIGS. 7a, 7 b, 8 a, 8 b, 9 a, 9 b, 10 a and 10 b.
 21. A methodfor preventing or treating RSV infection in susceptible or RSV infectedpersons which comprises administering a prophylactically ortherapeutically effective amount of one or more human monoclonalantibodies which possess an affinity to the RSV F-protein of ≦2×10⁻⁹molar and which also neutralize RSV in vitro.
 22. The method of claim 21wherein said antibodies are selected from the group consisting of RF-1,RF-2 and recombinant human monoclonal antibodies which contain thevariable heavy and light domains of RF-1 or RF-2.
 23. The method ofclaim 21 wherein said antibodies are administered by injection or byaerosol.
 24. The method of claim 21 wherein said antibodies areadministered in combination with an adjuvant.
 25. The method of claim 24wherein said adjuvant is Complete Freund's Adjuvant (CFA), Alum or acombination thereof.
 26. A pharmaceutical composition suitable forpreventing or treating RSV infection in susceptible or RSV infectedpersons which comprises a prophylactically or therapeutically effectiveamount of human monoclonal antibodies which neutralize RSV in vitro andwhich possess an affinity for the RSV F-protein of ≦2×10⁻⁹ molar and apharmaceutically acceptable carrier.
 27. The pharmaceutical compositionof claim 26 wherein said human monoclonal antibodies are selected fromthe group consisting of RF-1, RF-2 and recombinant human monoclonalantibodies which contain the variable heavy and light domains of eitherRF-1 or RF-2.
 28. A method of detecting the presence of RSV in ananalyte which comprises incubating, said analyte with a human monoclonalantibody which possesses an affinity for the RSV F-protein of ≦2×10⁻⁹molar under conditions which provide for the formation of RSV F-proteinantibody immune complexes; and detecting the presence of said RSVF-protein antibody immune complexes to determine whether RSV is presentin the analyte.
 29. The method of claim 28 wherein said antibody is RF-1or RF-2.
 30. The method of claim 29 wherein said antibody is directly orindirectly attached to a reporter molecule.
 31. The method of claim 30wherein said reporter molecule is a detectable enzyme or radionuclide.32. The method of claim 28 wherein the analyte comprises fluid obtainedfrom respiratory tissue.
 33. A test kit for assaying the presence of RSVin an analyte which comprises: (i) a human monoclonal antibody having anaffinity for the RSV F-protein of ≦2×10⁻⁹ molar; and (ii) a reportermolecule which is directly or indirectly attached to said humanmonoclonal antibody.
 34. The test kit of claim 33 wherein said humanmonoclonal antibody is RF-1 or RF-2.